Further novel oligonucleotide-ligand conjugates

The present invention relates to a compound comprising a modified saccharide moiety conjugated to a nucleic acid. The compound is useful in medicine for RNA interference therapy or for research and diagnostic purposes. In particular, the compound is useful in treating liver disease.

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Description
FIELD OF THE INVENTION

The present invention relates to novel nucleic acid conjugate compounds. The invention further relates to compositions comprising said conjugates and their use in medicine, research and diagnostics. The novel conjugate compounds may be used in the treatment of many diseases including central-nervous-system diseases, inflammatory diseases, metabolic disorders, genetic and inherited diseases, oncology, infectious diseases, and ocular disease.

BACKGROUND OF THE INVENTION

Double-stranded RNA (dsRNA) has been shown to block gene expression (Fire et al., 1998 and Elbashir et al., 2001) and this has been termed RNA interference or “RNAi”, mediated by interfering RNA molecules (iRNA). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multi-component nuclease that destroys messenger RNAs homologous to the silencing trigger. iRNAs (interfering RNA) such as siRNA (short interfering RNA), antisense RNA, and micro-RNA are oligonucleotides that prevent the formation of proteins by gene-silencing i.e. inhibiting translation of the protein. Gene-silencing agents are becoming increasingly important for therapeutic applications in medicine.

Thus, means for efficient delivery of oligonucleotides, in particular double stranded siRNAs, to cells in vivo is becoming increasingly important and requires specific targeting and substantial protection from the extracellular environment, particularly serum proteins. One method of achieving specific targeting is to conjugate a targeting moiety to the iRNA duplex agent. The targeting moiety helps in targeting the iRNA duplex agent to the required target site and there is a need to design appropriate targeting moieties for the desired receptor sites for the conjugated molecules to be taken up by the cells such as by endocytosis.

For example, the Asialoglycoprotein receptor (ASGP-R) is a high capacity receptor, which is highly abundant on hepatocytes. One of the first disclosures of triantennary cluster glycosides was in U.S. Pat. No. 5,885,968. Conjugates having three GalNAc ligands and comprising phosphate groups are known and are described in Dubber et al. (2003). The ASGP-R shows a 50-fold higher affinity for N-Acetyl-D-Galactosylamine (GalNAc) than D-Gal.

Hepatocytes expressing the Lectin (asialoglycoprotein receptor; ASGPR), which recognizes specifically terminal β-galactosyl subunits of glycosylated proteins or other oligosaccharides (P. H. Weigel et. al., 2002) can be used for targeting a drug to the liver by covalent coupling of galactose or galactosamine to the drug substance (S. Ishibashi, et. al. 1994). Furthermore the binding affinity can be significantly increased by the multi-valency effect, which is achieved by the repetition of the targeting unit (E. A. L. Biessen et. al., 1995).

The ASGPR is a mediator for an active endosomal transport of terminal β-galactosyl containing glycoproteins, thus ASGPR is highly suitable for targeted delivery of drug candidates like siRNA, which have to be delivered into a cell (Akinc et al.).

However, targeting ligands developed so far do not always translate to in vivo setting and there is a clear need for more efficacious receptor specific ligand conjugated iRNA duplex agents and methods for their preparation for the in vivo delivery of oligonucleotide therapeutics, nucleic acids and double stranded siRNAs. The present invention attempts to address these needs.

SUMMARY OF THE INVENTION

The present invention relates to conjugate compounds having three saccharide ligands and modified phosphate groups within the saccharide portion of the compounds. These conjugate compounds have been shown to have improved potency and duration in vivo. In addition, the conjugate groups are much easier to prepare compared to conjugates known in the art.

The conjugate compounds of the present invention have the formula I:
[S—X1—P—X2]3-A-X3—Z  (I)
wherein:
S represents a saccharide;
X1 represents C3-C6 alkylene or (—CH2—CH2—O)m(—CH2)2— wherein m is 1, 2, or 3;
P is a modified phosphate;
X2 is C1-C8 alkylene;
X3 represents a bridging unit;
Z is a nucleic acid;
the linkage between X3 and Z is a phosphate or thiophosphate;
and A is a branching unit selected from:


wherein:
R1 is hydrogen or C1-C10 alkylene;
and R2 is C1-C10 alkylene.

The nucleic acid may be selected from RNAi, siRNA, siNA, antisense nucleic acid, ribozymes, aptamers and spiegelmers. The present invention also relates to pharmaceutical compositions comprising the conjugate compound of formula I.

DETAILED DESCRIPTION OF THE INVENTION

The definitions and explanations below are for the terms as used throughout this entire document including both the specification and the claims.

Unless specified otherwise, the following terms have the following meanings:

“Conjugate” or “conjugate group” means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamics, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.

“GalNAc” means N-acetyl galactosamine.

Cx-Cy alkyl refers to a saturated aliphatic hydrocarbon group having x-y carbon atoms which may be linear or branched. For example C1-C6 alkyl and includes C1, C2, C3, C4, C5 and C6. “Branched” means that at least one carbon branch point is present in the group. For example, tert-butyl and isopropyl are both branched groups. Examples of C1-C6 alkyl groups include methyl, ethyl, propyl, butyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3 methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl and n-hexyl. This also applies for C1-C6 alkylene.

Cx-Cy alkoxy refers to a group or part of a group having an —O—Cx-Cy alkyl group according to the definition of Cx-Cy alkyl above. C1-C3 alkoxy contains from 1 to 3 carbon atoms and includes C1, C2 and C3. Examples of C1-C3 alkoxy include methoxy, ethoxy, propoxy and isopropoxy. Alkoxy as employed herein also extends to embodiments in which the or an oxygen atom (e.g. a single oxygen atom) is located within the alkyl chain, for example CH2CH2OCH3 or CH2OCH3. Thus the alkoxy may be linked through carbon to the remainder of the molecule, for example, —CH2CH2OCH3, or alternatively, the alkoxy is linked through oxygen to the remainder of the molecule, for example —OC1-3 alkyl. In certain instances, the alkoxy may be linked through oxygen to the remainder of the molecule but the alkoxy group contains a further oxygen atom, for example —OCH2CH2OCH3.

The term “nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids (ssDNA), double-stranded nucleic acids (dsDNA), small interfering ribonucleic acids (siRNA) and microRNAs (miRNA). A nucleic acid may also comprise any combination of these elements in a single molecule.

The term “treat” or “treating” or “treatment” may include prophylaxis and means to ameliorate, alleviate symptoms, eliminate the causation of the symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of the named disorder or condition. The compounds of the invention are useful in the treatment of humans and non-human animals.

By “effective amount” or “therapeutically effective amount” or “effective dose” is meant that amount sufficient to elicit the desired pharmacological or therapeutic effects, thus resulting in effective prevention or treatment of the disorder. Prevention of the disorder is manifested by delaying the onset of the symptoms of the disorder to a medically significant extent. Treatment of the disorder is manifested by a decrease in the symptoms associated with the disorder or an amelioration of the reoccurrence of the symptoms of the disorder.

A “pharmaceutical composition” or “composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition can comprise one or more active agents and a pharmaceutical carrier e.g. a sterile aqueous solution.

In a first aspect, the present invention provides compounds of formula I:
[S—X1—P—X2]3-A-X3—Z  (I)
wherein:
S represents a saccharide;
X1 represents C3-C6 alkylene or (—CH2—CH2—O)m(—CH2)2— wherein m is 1, 2, or 3;
P is a modified phosphate;
X2 is C1-C8 alkylene;
X3 represents a bridging unit;
Z is a nucleic acid;
the linkage between X3 and Z is a phosphate or thiophosphate;
and A is a branching unit selected from:


wherein:
R1 is hydrogen or C1-C10 alkylene;
and R2 is C1-C10 alkylene.

Preferably, R2 is a linear C1-C10 alkylene.

Preferably, the branching unit A has the structure:


wherein:
R1 is hydrogen or C1-C10 alkylene;
R2 is C1-C10 alkylene.

The branching unit A may be selected from the following:


wherein X3 is attached to the nitrogen atom; or


wherein X3 is attached to the nitrogen atom.

The “X3” portion of the compounds of formula I is a bridging unit. X3 may also be referred to as the conjugate linker. The bridging unit is linear and is covalently bound to the branching unit and the nucleic acid.

X3 may be selected from —C1-C20 alkylene-, —C2-C20 alkenylene-, an alkylene ether of formula —(C1-C20 alkylene)-O—(C1-C20 alkylene)-, —C0-C4 alkylene(Cy)C0-C4 alkylene- wherein Cy represents a substituted or unsubstituted 5 or 6 membered cycloalkylene, arylene, heterocyclylene or heteroarylene ring, —C1-C4 alkylene-NHC(O)—C1-C4 alkylene-, —C1-C4 alkylene-C(O)NH—C1-C4 alkylene-, —C1-C4 alkylene-SC(O)—C1-C4 alkylene-, —C1-C4 alkylene-C(O)S—C1-C4 alkylene-, —C1-C4 alkylene-OC(O)—C1-C4 alkylene-, —C1-C4 alkylene-C(O)O—C1-C4 alkylene-, and —C1-C6 alkylene-S—S—C1-C6 alkylene-.

X3 may be C1-C20 alkylene. Preferably, X3 is selected from the group consisting of —C3H6—, —C4H8—, —C6H12— and —C8H16—, especially —C4H8—, —C6H12— and —C8H16—.

P represents a modified phosphate group. P can be represented by:


wherein Y1 and Y2 each independently represent ═O, ═S, —O−—, —OH, —SH, —BH3, —OCH2CO2, —OCH2CO2Rx, —OCH2C(S)ORx, and —ORx, wherein Rx represents C1-C6 alkyl and wherein


indicates attachment to the remainder of the compound.

For example, Y1 may represent —OH and Y2 may represent ═O or ═S; or

Y1 may represent —O− and Y2 may represent ═O or ═S;

Y1 may represent ═O and Y2 may represent —CH3, —SH, —ORx, or —BH3;

Y1 may represent ═S and Y2 may represent —CH3, ORx or —SH.

It will be understood by the skilled person that in certain instances there will be delocalisation between Y1 and Y2.

Preferably, the modified phosphate group is a thiophosphate group. Thiophosphate groups include bithiophosphate (i.e. where Y1 represents ═S and Y2 represents —S−) and monothiophosphate (i.e. where Y1 represents —O− and Y2 represents ═S, or where Y1 represents ═O and Y2 represents —S−). Preferably, P is a monothiophosphate. The inventors have found that conjugates having thiophosphate groups in replacement of phosphate groups have improved potency and duration of action in vivo.

P may also be an ethylphosphate (i.e. where Y1 represents ═O and Y2 represents OCH2CH3).

The saccharide, which can also be referred to as the ligand, may be selected to have an affinity for at least one type of receptor on a target cell. In particular, the receptor is on the surface of a mammalian liver cell, for example, the hepatic asialoglycoprotein receptor (ASGP-R).

For any of the above aspects, the saccharide may be selected from N-acetyl galactosamine, mannose, galactose, glucose, glucosamine and fructose. Preferably, the saccharide is N-acetyl galactosamine (GalNAc). The compounds of the invention have 3 ligands which are each preferably N-acetyl galactosamine.

“GalNAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetyl galactosamine. Reference to “GalNAc” or “N-acetyl galactosamine” includes both the β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and the α-form: 2-(Acetylamino)-2-deoxy-α-D-galactopyranose. In certain embodiments, both the β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form: 2-(Acetylamino)-2-deoxy-α-D-galactopyranose may be used interchangeably. Preferably, the compounds of the invention comprise the β-form, 2-(Acetylamino)-2-deoxy-β-D-galactopyranose.

The “X1—P—X2” portion of the compounds of the present invention may also be referred to as the tether or linker. The linker comprises a linear group and is covalently attached to the saccharide ligand and the branching unit.

X1 may be (—CH2—CH2—O)m(—CH2)2— wherein m is 1, 2, or 3. X1 may be (—CH2—CH2—O)(—CH2)2—. X1 may be (—CH2—CH2—O)2(—CH2)2—. X1 may be (—CH2—CH2—O)3(—CH2)2—. Preferably, X1 is (—CH2—CH2—O)2(—CH2)2—. Alternatively, X1 represents C3-C6 alkylene. X1 may be propylene. X1 may be butylene. X1 may be pentylene. X1 may be hexylene. Preferably the alkyl is a linear alkylene. In particular, X1 may be butylene.

In a second aspect, the present invention provides conjugate compounds having the structure:


wherein Z is a nucleic acid.

In a third aspect, the present invention provides conjugate compounds having the structure:


wherein Z is a nucleic acid.

In a fourth aspect, the present invention provides conjugate compounds having the structure:


wherein Z is a nucleic acid.

In a fifth aspect, the present invention provides conjugate compounds having the structure:


wherein Z is a nucleic acid.

In all cases described herein, the nucleic acid may be selected from the group consisting of DNA, RNA, PNA and LNA.

The nucleic acid may be a functional nucleic acid, whereby preferably the functional nucleic acid is selected from the group consisting of mRNA, micro-RNA, shRNA, combinations of RNA and DNA, siRNA, siNA, antisense nucleic acid, ribozymes, aptamers and spiegelmers. In particular, the nucleic acid may be siRNA.

The nucleic acid may be selected from RNAi, siRNA, antisense nucleic acid, ribozymes, aptamers and spiegelmers.

The nucleic acids may be of any length and can have any number of nucleotides such that they are effective for RNAi. Preferably, the siRNAs range from 15 to 30 nucleotides. The duplex region of a double stranded RNA may range from 15 to 30 nucleotide base pairs using the Watson-crick base pairing. The duplex region may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 base pairs.

Preferably, the nucleic acid has 19 to 23 base pairs. For example, the nucleic acid may be 19, 20, 21, 22 or 23 base pairs in length.

The double stranded iRNAs may be blunt ended at one end or on both ends. The double stranded iRNAs may have overhangs of 1 or more nucleotides one or both strands at one or both ends. The overhangs may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides n length.

For any of the above aspects, the nucleic acid may be a modified nucleic acid. The modification may be selected from substitutions or insertions with analogues of nucleic acids or bases and chemical modification of the base, sugar or phosphate moieties.

The nucleic acid may: a) be blunt ended at both ends; b) have an overhang at one end and a blunt end at the other; or c) have an overhang at both ends.

One or more nucleotides on the first and/or second strand may be modified, to form modified nucleotides. One or more of the odd numbered nucleotides of the first strand may be modified. One or more of the even numbered nucleotides of the first strand may be modified by at least a second modification, wherein the at least second modification is different from the modification on the one or more add nucleotides. At least one of the one or more modified even numbered nucleotides may be adjacent to at least one of the one or more modified odd numbered nucleotides.

A plurality of odd numbered nucleotides in the first strand may be modified in the nucleic acid of the invention. A plurality of even numbered nucleotides in the first strand may be modified by a second modification. The first strand may comprise adjacent nucleotides that are modified by a common modification. The first strand may also comprise adjacent nucleotides that are modified by a second different modification.

One or more of the odd numbered nucleotides of the second strand may be modified by a modification that is different to the modification of the odd numbered nucleotides on the first strand and/or one or more of the even numbered nucleotides of the second strand may be by the same modification of the odd numbered nucleotides of the first strand. At least one of the one or more modified even numbered nucleotides of the second strand may be adjacent to the one or more modified odd numbered nucleotides. A plurality of odd numbered nucleotides of the second strand may be modified by a common modification and/or a plurality of even numbered nucleotides may be modified by the same modification that is present on the first strand odd numbered nucleotides. A plurality of odd numbered nucleotides on the second strand may be modified by a second modification, wherein the second modification is different from the modification of the first strand odd numbered nucleotides.

The second strand comprises adjacent nucleotides that are modified by a common modification, which may be a second modification that is different from the modification of the odd numbered nucleotides of the first strand.

In the nucleic acid of the invention, each of the odd numbered nucleotides in the first strand and each of the even numbered nucleotides in the second strand may be modified with a common modification and, each of the even numbered nucleotides may be modified in the first strand with a second modification and each of the odd numbered nucleotides may be modified in the second strand with a second different modification.

The nucleic acid of the invention may have the modified nucleotides of the first strand shifted by at least one nucleotide relative to the unmodified or differently modified nucleotides of the second strand.

The modification and/or modifications may each and individually be selected from the group consisting of 3′ terminal deoxy thymine, 2′ O methyl, a 2′ deoxy modification, a 2′ amino modification, a 2′ alkyl modification, a morpholino modification, a phosphoramidate modification, 5′-phosphorothioate group modification, a 5′ phosphate or 5′ phosphate mimic modification and a cholesteryl derivative or a dodecanoic acid bisdecylamide group modification and/or the modified nucleotide may be any one of a locked nucleotide, an abasic nucleotide or a non natural base comprising nucleotide. At least one modification may be 2′-O-methyl and/or at least one modification may be 2′-F. By nucleic acid it is meant a nucleic acid comprising two strands comprising nucleotides, that is able to interfere with gene expression. Inhibition may be complete or partial and results in down regulation of gene expression in a targeted manner. The nucleic acid comprises two separate polynucleotide strands; the first strand, which may also be a guide strand; and a second strand, which may also be a passenger strand. The first strand and the second strand may be part of the same polynucleotide molecule that is self complementary which ‘folds’ to form a double stranded molecule. The nucleic acid may be an siRNA molecule.

The first strand may also be referred to as an antisense strand. The second strand may also be referred to as a sense strand.

The nucleic acid may comprise ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or nucleotide analogous. The nucleic acid may further comprise a double stranded nucleic acid portion or duplex region formed by all or a portion of the first strand (also known in the art as a guide strand) and all or a portion of the second strand (also known in the art as a passenger strand). The duplex region is defined as beginning with the first base pair formed between the first strand and the second strand and ending with the last base pair formed between the first strand and the second strand, inclusive.

By duplex region refers it is meant the region in two complementary or substantially complementary oligonucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary. For example, an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, yet only 19 nucleotides on each strand are complementary or substantially complementary, such that the “duplex region” consists of 19 base pairs. The remaining base pairs may exist as 5′ and 3′ overhangs, or as single stranded regions. Further, within the duplex region, 100% complementarity is not required; substantial complementarity is allowable within a duplex region. Substantial complementarity refers to complementarity between the strands such that they are capable of annealing under biological conditions. Techniques to empirically determine if two strands are capable of annealing under biological conditions are well known in the art. Alternatively, two strands can be synthesised and added together under biological conditions to determine if they anneal to one another.

The portion of the first strand and second strand that form at least one duplex region may be fully complementary and are at least partially complementary to each other. Depending on the length of an nucleic acid, a perfect match in terms of base complementarity between the first strand and second strand is not necessarily required. However, the first and second strands must be able to hybridise under physiological conditions.

The complementarity between the first strand and second strand in the at least one duplex region may be perfect in that there are no nucleotide mismatches or additional/deleted nucleotides in either strand. Alternatively, the complementarity may not be perfect. The complementarity may be at least 70%, 75%, 80%, 85%, 90% or 95%.

The first strand and the second strand may each comprise a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides.

An “overhang” as used herein has its normal and customary meaning in the art, i.e. a single stranded portion of a nucleic acid that extends beyond the terminal nucleotide of a complementary strand in a double strand nucleic acid. The term “blunt end” includes double stranded nucleic acid whereby both strands terminate at the same position, regardless of whether the terminal nucleotide(s) are base paired. The terminal nucleotide of a first strand and a second strand at a blunt end may be base paired. The terminal nucleotide of a first strand and a second strand at a blunt end may not be paired. The terminal two nucleotides of an first strand and a second strand at a blunt end may be base paired. The terminal two nucleotides of an first strand and a second strand at a blunt end may not be paired.

The nucleic acid may have an overhang at one end and a blunt end at the other. The nucleic acid may have an overhang at both ends. The nucleic acid may be blunt ended at both ends. The nucleic acid may be blunt ended at the end with the 5′ end of the first strand and the 3′ end of the second strand or at the 3′-end of the first strand and the 5′ end of the second strand.

The nucleic acid may comprise an overhang at a 3′ or 5′ end. The nucleic acid may have a 3′ overhang on the first strand. The nucleic acid may have a 3′ overhang on the second strand. The nucleic acid may have a 5′ overhang on the first strand. The nucleic acid may have a 5′ overhang on the second strand. The nucleic acid may have an overhang at both the 5′ end and 3′ end of the first strand. The nucleic acid may have an overhang at both the 5′ end and 3′ end of the second strand. The nucleic acid may have a 5′ overhang on the first strand and a 3′ overhang on the second strand. The nucleic acid may have a 3′ overhang on the first strand and a 5′ overhang on the second strand. The nucleic acid may have a 3′ overhang on the first strand and a 3′ overhang on the second strand. The nucleic acid may have a 5′ overhang on the first strand and a 5′ overhang on the second strand.

An overhang at the 3′-end or 5′ end of the second strand or the first strand may be selected from consisting of 1, 2, 3, 4 and 5 nucleotides in length. Optionally, an overhang may consist of 1 or 2 nucleotides, which may or may not be modified.

Unmodified polynucleotides, particularly ribonucleotides, may be prone to degradation by cellular nucleases, and, as such, modification/modified nucleotides may be included in the nucleic acid of the invention.

One or more nucleotides on the second and/or first strand of the nucleic acid of the invention may be modified.

Modifications of the nucleic acid of the present invention generally provide a powerful tool in overcoming potential limitations including, but not limited to, in vitro and in vivo stability and bioavailability inherent to native RNA molecules. The nucleic acid according to the invention may be modified by chemical modifications. Modified nucleic acid can also minimise the possibility of inducing interferon activity in humans. Modification can further enhance the functional delivery of a nucleic acid to a target cell. The modified nucleic acid of the present invention may comprise one or more chemically modified ribonucleotides of either or both of the first strand or the second strand. A ribonucleotide may comprise a chemical modification of the base, sugar or phosphate moieties. The ribonucleic acid may be modified by substitution or insertion with analogues of nucleic acids or bases.

One or more nucleotides of a nucleic acid of the present invention may be modified. The nucleic acid may comprise at least one modified nucleotide. The modified nucleotide may be on the first strand. The modified nucleotide may be in the second strand. The modified nucleotide may be in the duplex region. The modified nucleotide may be outside the duplex region, i.e., in a single stranded region. The modified nucleotide may be on the first strand and may be outside the duplex region. The modified nucleotide may be on the second strand and may be outside the duplex region. The 3′-terminal nucleotide of the first strand may be a modified nucleotide. The 3′-terminal nucleotide of the second strand may be a modified nucleotide. The 5′-terminal nucleotide of the first strand may be a modified nucleotide. The 5′-terminal nucleotide of the second strand may be a modified nucleotide.

An nucleic acid of the invention may have 1 modified nucleotide or a nucleic acid of the invention may have about 2-4 modified nucleotides, or a nucleic acid may have about 4-6 modified nucleotides, about 6-8 modified nucleotides, about 8-10 modified nucleotides, about 10-12 modified nucleotides, about 12-14 modified nucleotides, about 14-16 modified nucleotides about 16-18 modified nucleotides, about 18-20 modified nucleotides, about 20-22 modified nucleotides, about 22-24 modified nucleotides, 24-26 modified nucleotides or about 26-28 modified nucleotides. In each case the nucleic acid comprising said modified nucleotides retains at least 50% of its activity as compared to the same nucleic acid but without said modified nucleotides. The nucleic acid may retain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or above of its activity as compared to the same nucleic acid but without said modified nucleotides

The modified nucleotide may be a purine or a pyrimidine. At least half of the purines may be modified. At least half of the pyrimidines may be modified. All of the purines may be modified. All of the pyrimidines may be modified. The modified nucleotides may be selected from the group consisting of a 3′ terminal deoxy thymine (dT) nucleotide, a 2′ O methyl modified nucleotide, a 2′ modified nucleotide, a 2′ deoxy modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′ amino modified nucleotide, a 2′ alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non natural base comprising nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a nucleotide comprising a 5′ phosphate or 5′ phosphate mimic and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

The nucleic acid may comprise a nucleotide comprising a modified nucleotide, wherein the base is selected from 2-aminoadenosine, 2,6-diaminopurine,inosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), 6-azapyrimidine, 6-alkylpyrimidine (e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid and 2-thiocytidine.

Nucleic acids discussed herein include unmodified RNA as well as RNA which have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, for example as occur naturally in the human body. Modified nucleotide as used herein refers to a nucleotide in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature. While they are referred to as modified nucleotides they will of course, because of the modification, include molecules which are not nucleotides, for example a polynucleotide molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows hybridisation between strands i.e. the modified nucleotides mimic the ribophosphate backbone.

Many of the modifications described below that occur within a nucleic acid will be repeated within a polynucleotide molecule, such as a modification of a base, or a phosphate moiety, or the a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the possible positions/nucleotides in the polynucleotide but in many cases it will not. A modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal regions, such as at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an nucleic acid of the invention or may only occur in a single strand region of an nucleic acid of the invention. A phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4 or 5 nucleotides of a strand, or may occur in duplex and/or in single strand regions, particularly at termini. The 5′ end or 3′ ends may be phosphorylated.

Stability of an nucleic acid of the invention may be increased by including particular bases in overhangs, or to include modified nucleotides, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. Purine nucleotides may be included in overhangs. All or some of the bases in a 3′ or 5′ overhang may be modified. Modifications can include the use of modifications at the 2′ OH group of the ribose sugar, the use of deoxyribonucleotides, instead of ribonucleotides, and modifications in the phosphate group, such as phosphothioate modifications. Overhangs need not be homologous with the target sequence.

Nucleases can hydrolyze nucleic acid phosphodiester bonds. However, chemical modifications to nucleic acids can confer improved properties, and, can render oligoribonucleotides more stable to nucleases.

Modified nucleic acids, as used herein, can include one or more of:

(i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens (referred to as linking even if at the 5′ and 3′ terminus of the nucleic acid of the invention);

(ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar;

(iii) replacement of the phosphate moiety with “dephospho” linkers;

(iv) modification or replacement of a naturally occurring base;

(v) replacement or modification of the ribose-phosphate backbone;

(vi) modification of the 3′ end or 5′ end of the RNA, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, e.g., a fluorescently labeled moiety, to either the 3′ or 5′ end of RNA.

The terms replacement, modification, alteration, indicates a difference from a naturally occurring molecule.

Specific modifications are discussed in more detail below.

Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulphur. One, each or both non-linking oxygens in the phosphate group can be independently any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl).

The phosphate linker can also be modified by replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen. Replacement of the non-linking oxygens with nitrogen is possible.

A modified nucleotide can include modification of the sugar groups. The 2′ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR; “locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE (AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy, O(CH2)nAMINE, (e.g., AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino).

“Deoxy” modifications include hydrogen halo; amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH2CH2NH)nCH2CH2-AMINE (AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino),

—NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality. Other substitutents of certain embodiments include 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and 2′-fluoro.

The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleotides may contain a sugar such as arabinose.

Modified nucleotides can also include “abasic” sugars, which lack a nucleobase at C—I′. These abasic sugars can further contain modifications at one or more of the constituent sugar atoms.

The 2′ modifications may be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate).

The phosphate group can be replaced by non-phosphorus containing connectors.

Examples of moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. In certain embodiments, replacements may include the methylenecarbonylamino and methylenemethylimino groups.

The phosphate linker and ribose sugar may be replaced by nuclease resistant nucleotides. Examples include the mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. In certain embodiments, PNA surrogates may be used.

The 3′ and 5′ ends of an oligonucleotide can be modified. Such modifications can be at the 3′ end or the 5′ end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group. For example, the 3′ and 5′ ends of an oligonucleotide can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester). The functional molecular entities can be attached to the sugar through a phosphate group and/or a linker. The terminal atom of the linker can connect to or replace the linking atom of the phosphate group or the C-3′ or C-5′O, N, S or C group of the sugar. Alternatively, the linker can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., —(CH2)n-, —(CH2)nN—, —(CH2)nO—, —(CH2)nS—, O(CH2CH2O)nCH2CH2OH (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. The 3′ end can be an —OH group.

Other examples of terminal modifications include dyes, intercalating agents (e.g., acridines), cross-linkers (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, including to modulate activity or to modulate resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5′ end with phosphate or phosphate analogs. Nucleic acids of the invention, on the first or second strand, may be 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)O-5′); 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g., 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-vinylphosphonate, 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O) 0-5′-).

The nucleic acid of the present invention may include one or more phosphorothioate modifications on one or more of the terminal ends of the first and/or the second strand. Optionally, each or either end of the first strand may comprise one or two or three phosphorothioate modified nucleotides. Optionally, each or either end of the second strand may comprise one or two or three phosphorothioate modified nucleotides. Optionally, both ends of the first strand and the 5′ end of the second strand may comprise two phosphorothioate modified nucleotides. By phosphorothioate modified nucleotide it is meant that the linkage between the nucleotide and the adjacent nucleotide comprises a phosphorothioate group instead of a standard phosphate group.

Terminal modifications can also be useful for monitoring distribution, and in such cases the groups to be added may include fluorophores, e.g., fluorscein or an Alexa dye. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-linking an RNA agent to another moiety.

Adenine, guanine, cytosine and uracil are the most common bases found in RNA. These bases can be modified or replaced to provide RNA's having improved properties. E.g., nuclease resistant oligoribonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the above modifications. Alternatively, substituted or modified analogs of any of the above bases and “universal bases” can be employed. Examples include 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N<4>-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.

As used herein, the terms “non-pairing nucleotide analog” means a nucleotide analog which includes a non-base pairing moiety including but not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me riboT, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC. In some embodiments the non-base pairing nucleotide analog is a ribonucleotide. In other embodiments it is a deoxyribonucleotide.

As used herein, the term, “terminal functional group” includes without limitation a halogen, alcohol, amine, carboxylic, ester, amide, aldehyde, ketone, ether groups.

Certain moieties may be linked to the 5′ terminus of the first strand or the second strand and includes abasic ribose moiety, abasic deoxyribose moiety, modifications abasic ribose and abasic deoxyribose moieties including 2′ alkyl modifications; inverted abasic ribose and abasic deoxyribose moieties and modifications thereof, C6-imino-Pi; a mirror nucleotide including L-DNA and L-RNA; 5′OMe nucleotide; and nucleotide analogs including 4′,5′-methylene nucleotide; 1-(β-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; alpha-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted abasic moiety; 1,4-butanediol phosphate; 5′-amino; and bridging or non bridging methylphosphonate and 5′-mercapto moieties.

The nucleic acids of the invention may be included one or more inverted nucleotides, for example inverted thymidine or inverted adenine (for example see Takei, et al., 2002. JBC 277 (26):23800-06).

As used herein, the term “inhibit”, “down-regulate”, or “reduce” with respect to gene expression means the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits (e.g., mRNA), or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of a nucleic acid of the invention; for example the expression may be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less than that observed in the absence of an inhibitor.

The nucleic acid of the present invention may comprise an abasic nucleotide. The term “abasic” as used herein, refers to moieties lacking a base or having other chemical groups in place of a base at the 1′ position, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative.

The nucleic acid may comprise one or more nucleotides on the second and/or first strands that are modified. Alternating nucleotides may be modified, to form modified nucleotides.

Alternating as described herein means to occur one after another in a regular way. In other words, alternating means to occur in turn repeatedly. For example if one nucleotide is modified, the next contiguous nucleotide is not modified and the following contiguous nucleotide is modified and so on. One nucleotide may be modified with a first modification, the next contiguous nucleotide may be modified with a second modification and the following contiguous nucleotide is modified with the first modification and so on, where the first and second modifications are different.

One or more of the odd numbered nucleotides of the first strand of the nucleic acid of the invention may be modified wherein the first strand is numbered 5′ to 3′. The term “odd numbered” as described herein means a number not divisible by two. Examples of odd numbers are 1, 3, 5, 7, 9, 11 and so on. One or more of the even numbered nucleotides of the first strand of the nucleic acid of the invention may be modified, wherein the first strand is numbered 5′ to 3′. The term “even numbered” as described herein means a number which is evenly divisible by two. Examples of even numbers are 2, 4, 6, 8, 10, 12, 14 and so on. One or more of the odd numbered nucleotides of the second strand of the nucleic acid of the invention may be modified wherein the second strand is numbered 3′ to 5′. One or more of the even numbered nucleotides of the second strand of the nucleic acid of the invention may be modified, wherein the second strand is numbered 3′ to 5′.

One or more nucleotides on the first and/or second strand may be modified, to form modified nucleotides. One or more of the odd numbered nucleotides of the first strand may be modified. One or more of the even numbered nucleotides of the first strand may be modified by at least a second modification, wherein the at least second modification is different from the modification on the one or more add nucleotides. At least one of the one or more modified even numbered nucleotides may be adjacent to at least one of the one or more modified odd numbered nucleotides.

A plurality of odd numbered nucleotides in the first strand may be modified in the nucleic acid of the invention. A plurality of even numbered nucleotides in the first strand may be modified by a second modification. The first strand may comprise adjacent nucleotides that are modified by a common modification. The first strand may also comprise adjacent nucleotides that are modified by a second different modification.

One or more of the odd numbered nucleotides of the second strand may be modified by a modification that is different to the modification of the odd numbered nucleotides on the first strand and/or one or more of the even numbered nucleotides of the second strand may be by the same modification of the odd numbered nucleotides of the first strand. At least one of the one or more modified even numbered nucleotides of the second strand may be adjacent to the one or more modified odd numbered nucleotides. A plurality of odd numbered nucleotides of the second strand may be modified by a common modification and/or a plurality of even numbered nucleotides may be modified by the same modification that is present on the first stand odd numbered nucleotides. A plurality of odd numbered nucleotides on the second strand may be modified by a second modification, wherein the second modification is different from the modification of the first strand odd numbered nucleotides.

The second strand may comprise adjacent nucleotides that are modified by a common modification, which may be a second modification that is different from the modification of the odd numbered nucleotides of the first strand.

In the nucleic acid of the invention, each of the odd numbered nucleotides in the first strand and each of the even numbered nucleotides in the second strand may be modified with a common modification and, each of the even numbered nucleotides may be modified in the first strand with a second modification and each of the odd numbered nucleotides may be modified in the second strand with the second modification.

The nucleic acid of the invention may have the modified nucleotides of the first strand shifted by at least one nucleotide relative to the unmodified or differently modified nucleotides of the second strand.

One or more or each of the odd numbered nucleotides may be modified in the first strand and one or more or each of the even numbered nucleotides may be modified in the second strand. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. One or more or each of the even numbered nucleotides may be modified in the first strand and one or more or each of the even numbered nucleotides may be modified in the second strand. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. One or more or each of the odd numbered nucleotides may be modified in the first strand and one or more of the odd numbered nucleotides may be modified in the second strand by a common modification. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. One or more or each of the even numbered nucleotides may be modified in the first strand and one or more or each of the odd numbered nucleotides may be modified in the second strand by a common modification. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification.

In a sixth aspect, the present invention provides a composition comprising a compound according to any of the above aspects and a suitable carrier or excipient.

In a seventh aspect, the present invention provides a compound according to any of the first to fifth aspects or a composition according to the sixth aspect for use in medicine.

The compound or composition may be for use in the treatment of liver diseases, genetic diseases, hemophilia and bleeding disorders, liver fibrosis, non-alcoholic steotohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), viral hepatitis, rare diseases (e.g. acromegaly), metabolic diseases (e.g. hypercholesterolemia, dyslipidemia, hypertriglyceridemia), cardiovascular diseases, obesity, thalassemia, liver injury (e.g. drug induced liver injury), hemochromatosis, alcoholic liver diseases, alcohol dependence, anemia, and anemia of chronic diseases.

The compound or composition may be for use in the treatment of liver disease, anemia, chronic diseases, Thalassemia; drug induced liver injury, hemochromatosis and anemia of chronic disease.

In an eighth aspect, the present invention provides a method of delivery of nucleic acids to hepatocytes comprising contacting the hepatocyte with a compound according to any of the first to fifth aspects. The method may be used in vitro or in vivo, for diagnostic purposes, therapy or research purposes.

In a ninth aspect, the present invention provides a process for making a compound of formula (I), according to any of the first to fifth aspects, the process comprising adding together each component to form the compound of formula (I).

Building blocks of the following structures may be used in the method of manufacture:


wherein DMTr=4,4′-dimethoxytrityl (DMTr).
RNA Modifications

Modifications of the siRNA molecules of the present invention generally provides a powerful tool in overcoming potential limitations including, but not limited to, in vitro and in vivo stability and bioavailability inherent to native RNA molecules. The siRNA according to the invention may be modified by chemical modifications. Modified siRNA can also minimize the possibility of activating interferon activity in humans. Modification can further enhance the functional delivery of a siRNA to a target cell. The modified siRNA of the present invention may comprise one or more chemically modified ribonucleotides of either or both of the antisense strand or the sense strand. A ribonucleotide may comprise a chemical modification of the base, sugar or phosphate moieties. The ribonucleic acid may be modified by substitution or insertion with analogues of nucleic acids or bases.

One or more nucleotides of a siRNA of the present invention may comprise a modified base. In one aspect, the siRNA comprises at least one nucleotide comprising a modified base. In one embodiment, the modified base in on the antisense strand. In another embodiment, the modified base in on the sense strand. In another embodiment, the modified base is in the duplex region. In another embodiment, the modified base is outside the duplex region, i.e., in a single stranded region. In another embodiment, the modified base is on the antisense strand and is outside the duplex region. In another embodiment, the modified base is on the sense strand and is outside the duplex region. In another embodiment, the 3′-terminal nucleotide of the antisense strand is a nucleotide with a modified base. In another embodiment, the 3′-terminal nucleotide of the sense strand is nucleotide with a modified base. In another embodiment, the 5′-terminal nucleotide of the antisense strand is nucleotide with a modified base. In another embodiment, the 5′-terminal nucleotide of the sense strand is nucleotide with a modified base.

In one embodiment, a siRNA may have 1 modified base. In another embodiment, a siRNA may have about 2-4 modified bases. In another embodiment, a siRNA has about 4-6 modified bases. In another embodiment, a siRNA has about 6-8 modified bases. In another embodiment, a siRNA has about 8-10 modified bases. In another embodiment, a siRNA has about 10-12 modified bases. In another embodiment, a siRNA has about 12-14 modified bases. In another embodiment, a siRNA has about 14-16 modified bases. In another embodiment, a siRNA has about 16-18 modified bases. In another embodiment, a siRNA has about 18-20 modified bases. In another embodiment, a siRNA has about 20-22 modified bases. In another embodiment, a siRNA has about 22-24 modified bases. In another embodiment, a siRNA has about 24-26 modified bases. In another embodiment, a siRNA has about 26-28 modified bases. In each case the siRNA comprising said modified bases retains at least 50% of its activity as compared to the same siRNA but without said modified bases.

The modified base may be a purine or a pyrimidine. In another embodiment, at least half of the purines are modified. In another embodiment, at least half of the pyrimidines are modified. In another embodiment, all of the purines are modified. In another embodiment, all of the pyrimidines are modified. In another embodiment, the siRNA may comprise a nucleotide comprising a modified base, wherein the base is selected from 2-aminoadenosine, 2,6-diaminopurine, inosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), 6-azapyrimidine, 6-alkylpyrimidine (e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid and 2-thiocytidine.

In another aspect, a siRNA of the present invention comprises an abasic nucleotide. The term “abasic” as used herein, refers to moieties lacking a base or having other chemical groups in place of a base at the 1′ position, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative. As used herein, a nucleotide with a modified base does not include abasic nucleotides. In one aspect, the siRNA comprises at least one abasic nucleotide. In one embodiment, the abasic nucleotide is on the antisense strand. In another embodiment, the abasic nucleotide is on the sense strand. In another embodiment, the abasic nucleotide is in the duplex region. In another embodiment, the abasic nucleotide is outside the duplex region. In another embodiment, the abasic nucleotide is on the antisense strand and is outside the duplex region. In another embodiment, the abasic nucleotide is on the sense strand and is outside the duplex region. In another embodiment, the 3′-terminal nucleotide of the antisense strand is an abasic nucleotide. In another embodiment, the 3′-terminal nucleotide of the sense strand is an abasic nucleotide. In another embodiment, the 5′-terminal nucleotide of the antisense strand is an abasic nucleotide. In another embodiment, the 5′-terminal nucleotide of the sense strand is an abasic nucleotide. In another embodiment, a siRNA has a number of abasic nucleotides selected from 1, 2, 3, 4, 5 and 6.

Modifications to Sugar Moiety

Another aspect relates to modifications to a sugar moiety. One or more nucleotides of a siRNA of the present invention may comprise a modified ribose moiety. Modifications at the 2′-position where the 2′-OH is substituted include the non-limiting examples selected from alkyl, substituted alkyl, alkaryl-, arylalkyl-, —F, —Cl, —Br, —CN, —CF3, —OCF3, —OCN, —O-alkyl, —S-alkyl, HS-alkyl-O, —O-alkenyl, —S-alkenyl, —N-alkenyl, —SO-alkyl, -alkyl-OSH,-alkyl-OH, —O-alkyl-OH, —O-alkyl-SH, —S-alkyl-OH, —S-alkyl-SH, -alkyl-S-alkyl, -alkyl-O-alkyl, —ONO2, —NO2, —N3, —NH2, alkylamino, dialkylamino-, aminoalkyl-, aminoalkoxy, aminoacid, aminoacyl-, —ONH2, —O-aminoalkyl, —O-aminoacid, —O-aminoacyl, heterocycloalkyl-, heterocycloalkaryl-, aminoalkylamino-, polyalklylamino-, substituted silyl-, methoxyethyl- (MOE), alkenyl and alkynyl. “Locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar is further included as a 2′ modification of the present invention. Preferred substituents are 2′-methoxyethyl, 2′-O—CH3, 2′-O-allyl, 2′-C-allyl, and 2′-fluoro. In one embodiment, the siRNA comprises 1-5 2′-modified nucleotides. In another embodiment, the siRNA comprises 5-10 2′-modified nucleotides. In another embodiment, the siRNA comprises 15-20 2′-modified nucleotides. In another embodiment, the siRNA comprises 20-25 2′-modified nucleotides. In another embodiment, the siRNA comprises 25-30 2′-modified nucleotides.

In one embodiment, the siRNA comprises 1-5 2′-O—CH3 modified nucleotides. In another embodiment, the siRNA comprises 5-10 2′-O—CH3 modified nucleotides. In another embodiment, the siRNA comprises 15-20 2′-O—CH3 modified nucleotides. In another embodiment, the siRNA comprises 20-25 2′-O—CH3 modified nucleotides. In another embodiment, the siRNA comprises 25-30 2′-O—CH3 modified nucleotides.

In one embodiment, the siRNA duplex region comprises 1-5 2′-O—CH3 modified nucleotides. In another embodiment, the siRNA duplex region comprises 5-10 2′-O—CH3 modified nucleotides. In another embodiment, the siRNA duplex region comprises 15-20 2′-O—CH3 modified nucleotides. In another embodiment, the siRNA duplex region comprises 20-25 2′-O—CH3 modified nucleotides. In another embodiment, the siRNA duplex region comprises 25-30 2′-O—CH3 modified nucleotides.

In one embodiment, the siRNA comprises an antisense strand of 19 nucleotides in length and a sense strand 19 nucleotides in length, wherein said antisense strand comprises 2′-O—CH3 modifications at nucleotides 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, and wherein said sense strand comprises 2′-O—CH3 modifications at nucleotides 2, 4, 6, 8, 10, 12, 14, 16 and 18, wherein said antisense strand is numbered from 5′-3′ and said sense strand is numbered from 3′-5′. In another embodiment, the siRNA comprises an antisense strand 20 nucleotides in length and a sense strand 20 nucleotides in length, wherein said antisense strand comprises 2′-O—CH3 modifications at nucleotides 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, and wherein said sense strand comprises 2′-O—CH3 modifications at nucleotides 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 wherein said antisense strand is numbered from 5′-3′ and said sense strand is numbered from 3′-5′.

In another embodiment, the siRNA comprises an antisense strand 21 nucleotides in length and a sense strand 21 nucleotides in length, wherein said antisense strand comprises 2′-O—CH3 modifications at nucleotides 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21, and wherein said sense strand comprises 2′-O—CH3 modifications at nucleotides 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, wherein said antisense strand is numbered from 5′-3′ and said sense strand is numbered from 3′-5′. In another embodiment, the siRNA comprises an antisense strand 22 nucleotides in length and a sense strand 22 nucleotides in length, wherein said antisense strand comprises 2′-O—CH3 modifications at nucleotides 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21, and wherein said sense strand comprises 2′-O—CH3 modifications at nucleotides 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22, wherein said antisense strand is numbered from 5′-3′ and said sense strand is numbered from 3′-5′. In another embodiment, the siRNA comprises an antisense strand 23 nucleotides in length and a sense strand 23 nucleotides in length, wherein said antisense strand comprises 2′-O—CH3 modifications at nucleotides 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23, and wherein said sense strand comprises 2′-O—CH3 modifications at nucleotides 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 wherein said antisense strand is numbered from 5′-3′ and said sense strand is numbered from 3′-5′.

In another embodiment, the siRNA comprises an antisense strand 18-23 nucleotides in length and a sense strand 18-23 nucleotides in length, wherein said antisense strand comprises 2′-O—CH3 modifications at nucleotides 3, 5, 7, 9, 11, 13, 15 and 17, and wherein said sense strand comprises 2′-O—CH3 modifications at nucleotides 4, 6, 8, 10, 12, 14 and 16, wherein said antisense strand is numbered from 5′-3′ and said sense strand is numbered from 3′-5′. In another embodiment, the siRNA comprises an antisense strand 18-23 nucleotides in length and a sense strand 18-23 nucleotides in length, wherein said antisense strand comprises 2′-O—CH3 modifications at nucleotides 5, 7, 9, 11, 13 and 15, and wherein said sense strand comprises 2′-O—CH3 modifications at nucleotides 6, 8, 10, 12 and 14, wherein said antisense strand is numbered from 5′-3′ and said sense strand is numbered from 3′-5′. In another embodiment, the siRNA comprises an antisense strand 18-23 nucleotides in length and a sense strand 18-23 nucleotides in length, wherein said antisense strand comprises 2′-O—CH3 modifications at nucleotides 7, 9, 11, 13 and wherein said sense strand comprises 2′-O—CH3 modifications at nucleotides 8, 10 and 12, wherein said antisense strand is numbered from 5′-3′ and said sense strand is numbered from 3′-5′. In another embodiment, the siRNA comprises an antisense strand 18-23 nucleotides in length and a sense strand 18-23 nucleotides in length, wherein said antisense strand comprises 2′-O—CH3 modifications at nucleotides 7, 9 and 11, and wherein said sense strand comprises 2′-O—CH3 modifications at nucleotides 8, 10 and 12, wherein said antisense strand is numbered from 5′-3′ and said sense strand is numbered from 3′-5′. In another embodiment, the siRNA comprises an antisense strand 18-23 nucleotides in length and a sense strand 18-23 nucleotides in length, wherein said antisense strand comprises 2′-O—CH3 modifications at nucleotides 7 and 9, and wherein said sense strand comprises 2′-O—CH3 modifications at nucleotides 8 and 10, wherein said antisense strand is numbered from 5′-3′ and said sense strand is numbered from 3′-5′. In another embodiment, the siRNA comprises an antisense strand 18-23 nucleotides in length and a sense strand 18-23 nucleotides in length, wherein said antisense strand comprises 2′-O—CH3 modifications at nucleotides 9 and 11, and wherein said sense strand comprises 2′-O—CH3 modifications at nucleotides 8 and 10, wherein said antisense strand is numbered from 5′-3′ and said sense strand is numbered from 3′-5′.

Pattern

In one aspect, the antisense duplex region comprises a plurality of groups of modified nucleotides, referred to herein as “modified groups”, wherein each modified group consists of one or more identically modified nucleotides, wherein each modified group is flanked on one or both sides by a second group of nucleotides, referred to herein as “flanking groups”, wherein each said flanking group consists of one or more nucleotides that are either unmodified or modified in a manner different from the nucleotides of said modified group. In one embodiment, each modified group in the antisense duplex region is identical, i.e., each modified group consists of an equal number of identically modified nucleotides. In another embodiment, each flanking group has an equal number of nucleotide. In another embodiment, each flanking group is identical. In another embodiment, the nucleotides of said modified groups in the antisense duplex region comprise a modified base. In another embodiment, the nucleotides of said modified groups comprise a modified phosphate backbone. In another embodiment, the nucleotides of said modified groups comprise a modified 2′ position.

In another aspect, the sense duplex region comprises a plurality of groups of modified groups, wherein each modified group consists of one or more identically modified nucleotides, wherein each modified group is flanked on one or both sides by a flanking group, wherein each said flanking group consists of one or more nucleotides that are either unmodified or modified in a manner different from the nucleotides of said modified group. In one embodiment, each modified group in the sense duplex region is identical. In another embodiment, each flanking group has an equal number of nucleotides. In another embodiment, each flanking group is identical. In another embodiment, the nucleotides of said modified groups in the sense duplex region comprise a modified base. In another embodiment, the nucleotides of said modified groups comprise a modified phosphate backbone. In another embodiment, the nucleotides of said modified groups comprise a modified 2′ position.

In another aspect, the antisense duplex region and the sense duplex region each comprise a plurality of modified groups, wherein each modified group consists of one or more identically modified nucleotides, wherein each modified group is flanked on one or both sides by a flanking group, wherein each said flanking group consists of one or more nucleotides that are either unmodified or modified in a manner different from the nucleotides of said modified group. In one embodiment, each modified group in the antisense duplex region and the sense duplex region are identical. In another embodiment, each flanking group in the antisense duplex region and the sense duplex region each have an equal number of nucleotides. In another embodiment, each flanking group in the antisense duplex region and in the sense duplex region are identical. In another embodiment, the nucleotides of said modified groups in the antisense duplex region and the sense duplex region each comprise the same modified groups and the same flanking groups. In another embodiment, the nucleotides of said modified groups in the antisense duplex region and the sense duplex region each comprise a modified base. In another embodiment, the nucleotides of said modified groups in the antisense duplex region and the sense duplex region each comprise a modified phosphate backbone. In another embodiment, the nucleotides of said modified groups in the antisense duplex region and the sense duplex region each comprise a modified 2′ position.

In one aspect, the antisense strand comprises a plurality of groups of modified nucleotides, referred to herein as “modified groups”, wherein each modified group consists of one or more identically modified nucleotides, wherein each modified group is flanked on one or both sides by a second group of nucleotides, referred to herein as “flanking groups”, wherein each said flanking group consists of one or more nucleotides that are either unmodified or modified in a manner different from the nucleotides of said modified group. In one embodiment, each modified group in the antisense strand is identical, i.e., each modified group consists of an equal number of identically modified nucleotides. In another embodiment, each flanking group has an equal number of nucleotide. In another embodiment, each flanking group is identical. In another embodiment, the nucleotides of said modified groups in the antisense strand comprise a modified base. In another embodiment, the nucleotides of said modified groups comprise a modified phosphate backbone. In another embodiment, the nucleotides of said modified groups comprise a modified 2′ position.

In another aspect, the sense strand comprises a plurality of groups of modified groups, wherein each modified group consists of one or more identically modified nucleotides, wherein each modified group is flanked on one or both sides by a flanking group, wherein each said flanking group consists of one or more nucleotides that are either unmodified or modified in a manner different from the nucleotides of said modified group. In one embodiment, each modified group in the sense strand is identical. In another embodiment, each flanking group has an equal number of nucleotides. In another embodiment, each flanking group is identical. In another embodiment, the nucleotides of said modified groups in the sense strand comprise a modified base. In another embodiment, the nucleotides of said modified groups comprise a modified phosphate backbone. In another embodiment, the nucleotides of said modified groups comprise a modified 2′ position.

In another aspect, the antisense strand and the sense strand each comprise a plurality of modified groups, wherein each modified group consists of one or more identically modified nucleotides, wherein each modified group is flanked on one or both sides by a flanking group, wherein each said flanking group consists of one or more nucleotides that are either unmodified or modified in a manner different from the nucleotides of said modified group. In one embodiment, each modified group in the antisense strand and the sense strand are identical. In another embodiment, each flanking group in the antisense strand and the sense strand each have an equal number of nucleotides. In another embodiment, each flanking group in the antisense strand and in the sense strand are identical. In another embodiment, the nucleotides of said modified groups in the antisense strand and the sense strand each comprise the same modified groups and the same flanking groups. In another embodiment, the nucleotides of said modified groups in the antisense strand and the sense strand each comprise a modified base. In another embodiment, the nucleotides of said modified groups in the antisense strand and the sense strand each comprise a modified phosphate backbone. In another embodiment, the nucleotides of said modified groups in the antisense strand and the sense strand each comprise a modified 2′ position.

In another aspect, the modified groups and the flanking groups form a regular pattern on the antisense stand. In another aspect, the modified groups and the flanking groups form a regular pattern on the sense strand. In one embodiment, the modified groups and the flanking groups form a regular pattern on the both the antisense strand and the sense strand. In another embodiment, the modified groups and the flanking groups form a regular pattern on the antisense duplex region. In another aspect, the modified groups and the flanking groups form a regular pattern on the sense duplex region. In one embodiment, the modified groups and the flanking groups form a regular pattern on the both the antisense duplex region and the sense duplex region.

In another aspect, the pattern is a spatial or positional pattern. A spatial or positional pattern means that (a) nucleotide(s) are modified depending on their position within the nucleotide sequence of a double-stranded portion. Accordingly, it does not matter whether the nucleotide to be modified is a pyrimidine or a purine. Rather the position of a modified nucleotide is dependent upon: (a) its numbered position on a strand of nucleic acid, wherein the nucleotides are numbered from the 5′-end to the 3′-end with the 5′-end nucleotide of the strand being position one (both the antisense strand and sense strand are numbered from their respective 5′-end nucleotide), or (b) the position of the modified group relative to a flanking group. Thus, according to this embodiment, the modification pattern will always be the same, regardless of the sequence which is to be modified.

In one embodiment, each modified group on both the antisense strand and the sense strand is identical. In one embodiment, each modified group on both the antisense duplex region and the sense duplex region is identical. In another embodiment, each modified group and each flanking group on both the antisense strand and the sense strand are identical. In one embodiment, each modified group and each flanking group on both the antisense duplex region and the sense duplex region are identical.

In one embodiment, each modified group, each modified group position, each flanking group and each flanking group position on both the antisense strand and the sense strand are identical. In one embodiment, each modified group, each modified group position, each flanking group and each flanking group position on both the antisense duplex region and the sense duplex region are identical. In another embodiment, the modified groups on the antisense strand are complementary with the modified groups on the sense strand (the modified groups on the antisense strand and the sense strand are perfectly aligned across from one another). In another embodiment, there are no mismatches in the modified groups such that each modified group on the antisense strand is base paired with each modified group on the sense strand.

In another embodiment, each modified group on the sense strand is shifted by 1, 2, 3, 4 or 5 nucleotides relative to the modified groups on the antisense strand. For example, if each modified group on the sense strand is shifted by one nucleotide or one group of nucleotides and a modified group started at position one on the antisense strand, a modified group on the sense strand would begin at position two. In another embodiment, the modified groups of the antisense strand do not overlap the modified groups of the sense strand, i.e., no nucleotide of a modified group on the antisense strand is base paired with a nucleotide of a modified group on the sense strand.

In one embodiment, deoxyribonucleotides at an end of a strand of nucleic acid are not considered when determining a position of a modified group, i.e., the positional numbering begins with the first ribonucleotide or modified ribonucleotide. In another embodiment, abasic nucleotides at an end of a strand of nucleic acid are not considered when determining a position of a modified group.

In one aspect, a modified group comprises a 5′-end nucleotide of either or both of the antisense strand and the sense strand. In another embodiment, a flanking group comprises the 5′-end nucleotide of either or both of the antisense strand and the sense strand. In another embodiment, the 5′-end nucleotide of either or both of the antisense strand and the sense strand is unmodified. In another embodiment, a modified group comprises the 5′-most nucleotide of either or both of the antisense duplex region and sense duplex region. In another embodiment, a flanking group comprises the 5′-most nucleotide of either or both of the antisense duplex region or the sense duplex region. In another embodiment, the 5′-most nucleotide of either or both of the antisense duplex region or the sense duplex region is unmodified. In one embodiment, the modification at the 2′ position is selected from the group comprising amino, fluoro, methoxy, alkoxy and C1-C3-alkyl. In another embodiment, the modification may be selected from 2′-O-methyl,2′-amino-2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-alkyl, and 2′-O—(C1-C3-alkyl). In another embodiment, the modification at the 2′ position is 2′-O-methyl.

In another aspect, each modified group consists of one nucleotide and each flanking group consists of one nucleotide. In one embodiment, each modified group on the antisense strand is aligned with a flanking group on the sense strand. In another embodiment, the alignment of each modified group on the antisense strand with the modified group on the sense strand is shifted by one or more nucleotides.

Modifications to Phosphate Backbone

Another aspect relates to modifications to a phosphate backbone. All or a portion of the nucleotides of the siRNA of the invention may be linked through phosphodiester bonds, as found in unmodified nucleic acid. A siRNA of the present invention however, may comprise a modified phosphodiester linkage. The phosphodiester linkages of either the antisense stand or the sense strand may be modified to independently include at least one heteroatom selected from nitrogen and sulfur. In one embodiment, a phosphoester group connecting a ribonucleotide to an adjacent ribonucleotide is replaced by a modified group. In one embodiment, the modified group replacing the phosphoester group is selected from phosphorothioate, methylphosphonate, phosphorodithioate or phosphoramidate group.

In one embodiment, all of the nucleotides of the antisense strand are linked through phosphodiester bonds. In another embodiment, all of the nucleotides of the antisense duplex region are linked through phosphodiester bonds. In another embodiment, all of the nucleotides of the sense strand are linked through phosphodiester bonds. In another embodiment, all of the nucleotides of the sense duplex region are linked through phosphodiester bonds. In another embodiment, the antisense strand comprises a number of modified phosphoester groups selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the antisense duplex region comprises a number of modified phosphoester groups selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the sense strand comprises a number of modified phosphoester groups selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the sense duplex region comprises a number of modified phosphoester groups selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

5′ and 3′ End Modifications

The siRNA of the present invention may include nucleic acid molecules comprising one or more modified nucleotides, abasic nucleotides, acyclic or deoxyribonucleotide at the terminal 5′- or 3′-end on either or both of the sense or antisense strands. In one embodiment, the 5′- and 3′-end nucleotides of both the sense and antisense strands are unmodified. In another embodiment, the 5′-end nucleotide of the antisense strand is modified. In another embodiment, the 5′-end nucleotide of the sense strand is modified. In another embodiment, the 3′-end nucleotide of the antisense strand is modified. In another embodiment, the 3′-end nucleotide of the sense strand is modified. In another embodiment, the 5′-end nucleotide of the antisense strand and the 5′-end nucleotide of the sense strand are modified. In another embodiment, the 3′-end nucleotide of the antisense strand and the 3′-end nucleotide of the sense strand are modified. In another embodiment, the 5′-end nucleotide of the antisense strand and the 3′-end nucleotide of the sense strand are modified. In another embodiment, the 3′-end nucleotide of the antisense strand and the 5′-end nucleotide of the sense strand are modified. In another embodiment, the 3′-end nucleotide of the antisense strand and both the 5′- and 3′-end nucleotides of the sense strand are modified. Both the 5′- and 3′-end nucleotides of the antisense strand may be modified. In another embodiment, both the 5′- and 3′-end nucleotides of the sense strand are modified.

The 5′-end nucleotide of the antisense strand may be phosphorylated. In another embodiment, the 5′-end nucleotide of the sense strand is phosphorylated. In another embodiment, the 5′-end nucleotides of both the antisense strand and the sense strand are phosphorylated. In another embodiment, the 5′-end nucleotide of the antisense strand is phosphorylated and the 5′-end nucleotide of the sense strand has a free hydroxyl group (5′-OH). In another embodiment, the 5′-end nucleotide of the antisense strand is phosphorylated and the 5′-end nucleotide of the sense strand is modified. In another embodiment the 5′-end nucleotide of the antisense strand carries a 5′E vinylphosphonate.

Modifications to the 5′- and 3′-end nucleotides are not limited to the 5′ and 3′ positions on these terminal nucleotides. Examples of modifications to end nucleotides include, but are not limited to, biotin, inverted (deoxy) abasics, amino, fluoro, chloro, bromo, CN, CF, methoxy, imidazole, carboxylate, thioate, C1 to C10 lower alkyl, substituted lower alkyl, alkaryl or arylalkyl, OCF3, OCN, O—, S—, or N-alkyl; O—, S—, or N-alkenyl; SO—CH3; SO2CH3; ONO2; NO2, N3; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino or substituted silyl, as, among others, described, e.g., in PCT patent application WO 99/54459, European patents EP 0586 520 B1 or EP 0618 925 B1, incorporated by reference in their entireties. As used herein, “alkyl” means C1-C12-alkyl and “lower alkyl” means C1-C6-alkyl, including C1-, C2-, C3-, C4-, C5- and C6-alkyl.

In another aspect, the 5′-end of the antisense strand, the 5′-end of the sense strand, the 3′-end of the antisense strand or the 3′-end of the sense strand may be covalently connected to a prodrug moiety. In one embodiment, the moiety may be cleaved in an endosome. In another the moiety may be cleaved in the cytoplasm.

Examples of different kinds of end modification(s) are presented in Table 1.

TABLE 1 Examples of end modifications Antisense strand Sense strand 1. 5′-end free OH free OH 3′-end free OH free OH 2. 5′-end free OH free OH 3′-end end modification end modification 3. 5′-end free OH free OH 3′-end free OH end modification 4. 5′-end free OH free OH 3′-end end modification free OH 5. 5′-end free OH end modification 3′-end free OH free OH 6. 5′-end free OH end modification 3′-end end modification free OH 7. 5′-end free OH end modification 3′-end free OH end modification 8. 5′-end free OH end modification 3′-end end modification end modification

In another embodiment, the terminal 3′ nucleotide or two terminal 3′-nucleotides on either or both of the antisense strand or sense strand is a 2′-deoxynucleotide. In another embodiment, the 2′-deoxynucleotide is a 2′-deoxy-pyrimidine. In another embodiment, the 2′-deoxynucleotide is a 2′ deoxy-thymidine.

shRNA (Short Hairpin Loop RNA) and Linked siRNA

Another aspect relates to shRNA and linked siRNA. The antisense strand and the sense strand may be covalently linked to each other. Such linkage may occur between any of the nucleotides forming the antisense strand and sense strand, respectively and can be formed by covalent or non-covalent linkages. Covalent linkage may be formed by linking both strands one or several times and at one or several positions, respectively, by a compound preferably selected from the group comprising methylene blue and bifunctinoal groups. Such bifunctional groups are preferably selected from the group comprising bis(2-chloroethyl)amine, N-acetly-N′-(p-glyoxylbenzoyl)cystamine, 4-thiouracile and psoralene.

Further, the antisense strand and the sense strand may be linked by a loop structure. The loop structure may be comprised of a non-nucleic acid polymer such as polyethylene glycol. The 5′-end of the antisense strand may be linked to the 3′-terminus of the sense strand or The 3′-end of the antisense strand may be linked to the 5′-end of the sense strand. The loop may consists of a nucleic acid, a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or the loop may be formed by polymers The length of the loop may be sufficient for linking the two strands covalently in a manner that a back folding can occur through a loop structure or similar structure.

The ribonucleic acid constructs may be incorporated into suitable vector systems. Preferably the vector comprises a promoter for the expression of RNAi. The promoter may be selected from any known in the art such as pol III, U6, H1 or 7SK.

The nucleic acids according to the present invention may comprise one or more phosphorothioate internucleotide linkage. Preferably the phosphorothioate internucleotide linkages may be distributed across the entire nucleotide sequences and may occur in any number at any position. Preferably the nucleic acids can comprise between one to ten phosphorothioate internucleotide linkages. Preferably the antisense strand has at least 1 phosphorothioate modification at each end. Preferably the antisense strand has 1-3 phosphorothioate modification at each end. Most preferably the antisense strand has 2 phosphorothioate modification at each end. Preferably the sense strand has at least 1 phosphorothioate modification at the 3′ end. Preferably the sense strand has 1-3 phosphorothioate modification at the 3′ end. Most preferably the sense strand has 2 phosphorothioate modifications at the 3′ end.

siRNA with Overhangs

An overhang at the 3′-end or 5′ end of the sense strand or the antisense strand may be selected from consisting of 1, 2, 3, 4 and 5 nucleotides in length. Alternatively, the siRNA molecule may be blunt-ended on both ends and may have a length of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 consecutive nucleotides.

In one embodiment, the siRNA molecule is blunt-ended on one end and the double stranded or douplex portion of the siRNA molecule has a length selected from 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 consecutive nucleotides.

In one embodiment, the siRNA molecule has overhangs on both ends on either strand and the double stranded or duplex portion of the siRNA molecule has a length of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 consecutive nucleotides.

The overhang may comprise at least one deoxyribonucleotides and/or a TT dinucleotide. Manufacture of the nucleic acid molecules of the present invention

The nucleic acid of the present invention can be produced using routine methods in the art including chemically synthesis or expressing the nucleic acid either in vitro (e.g., run off transcription) or in vivo. For example, using solid phase chemical synthesis or using an expression vector. In one embodiment, the expression vector can produce the nucleic acid of the invention in a target cell. Methods for the synthesis of the nucleic acid molecule described herein are known to persons skilled in the art.

Formulations for Delivery of the Nucleic Acids of the Present Invention

siRNAs and conjugated compounds can be delivered to cells, both in vitro and in vivo, by a variety of methods known to those skilled in the art, including direct contact with cells (“naked” siRNA) or by combination with one or more agents that facilitate targeting or delivery into cells. Such agents and methods include lipoplexes, liposomes, iontophoresis, hydrogels, cyclodextrins, nanocapsules, micro- and nanospheres and proteinaceous vectors. The nucleic acid/vehicle combination may be locally delivered in vivo by direct injection or by use of an infusion pump. The siRNA and conjugates of the invention can be delivered in vivo by various means including intravenous subcutaneous, intramuscular or intradermal injection or inhalation. The molecules can be used as pharmaceutical agents. Preferably, pharmaceutical agents prevent, modulate the occurrence, treat or alleviate a symptom of a disease state in a subject.

There is also provided the use of a composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing stability of a liposome or lipoplex solutions by preventing their aggregation and fusion. The formulations also have the added benefit in vivo of resisting opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug. Such liposomes have been may accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24780; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392). Long-circulating liposomes also protect the siRNA from nuclease degradation.

The siRNA conjugates of the present invention may be formulated as pharmaceutical compositions. The pharmaceutical compositions may be used as medicaments or as diagnostic agents, alone or in combination with other agents. For example, one or more siRNA conjugates of the invention can be combined with a delivery vehicle (e.g., liposomes) and excipients, such as carriers, diluents. Other agents such as preservatives and stabilizers can also be added. Methods for the delivery of nucleic acid molecules are known in the art and within the knowledge of the person skilled in the art.

The siRNA conjugates of the present invention can also be administered in combination with other therapeutic compounds, either administrated separately or simultaneously, e.g., as a combined unit dose. In one embodiment, the invention includes a pharmaceutical composition comprising one or more siRNA conjugates according to the present invention in a physiologically/pharmaceutically acceptable excipient, such as a stabilizer, preservative, diluent, buffer, and the like.

Dosage

Dosage levels for the medicament and pharmaceutical compositions of the invention can be determined by those skilled in the art by routine experimentation. In one embodiment, a unit dose may contain between about 0.01 mg/kg and about 100 mg/kg body weight of siRNA. Alternatively, the dose can be from 10 mg/kg to 25 mg/kg body weight, or 1 mg/kg to 10 mg/kg body weight, or 0.05 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to 1 mg/kg body weight, or 0.1 mg/kg to 0.5 mg/kg body weight, or 0.5 mg/kg to 1 mg/kg body weight.

The pharmaceutical composition may be a sterile injectable aqueous suspension or solution, or in a lyophilized form. In one embodiment, the pharmaceutical composition may comprise lyophilized lipoplexes or an aqueous suspension of lipoplexes. The lipoplexes preferably comprises a siRNA of the present invention. Such lipoplexes may be used to deliver the siRNA of the invention to a target cell either in vitro or in vivo.

The pharmaceutical compositions and medicaments of the present invention may be administered to a mammalian subject in a pharmaceutically effective dose. The mammal may be selected from humans, dogs, cats, horses, cattle, pig, goat, sheep, mouse, rat, hamster and guinea pig.

Routes of Delivery

A conjugated composition that includes a double stranded siRNA can be delivered to a subject by a variety of routes. Exemplary routes include: sub cutaneous, intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.

The conjugated composition can be incorporated into pharmaceutical compositions suitable for administration with a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the iRNA in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the iRNA and mechanically introducing the DNA.

It will be appreciated by one skilled in the art that the modification, modifications of the sugar moiety, pattern, 5′ and 3′ end modifications, overhangs, formulations, delivery, dosage and routes of delivery as described above may equally be applied to any type of iRNA molecule and is not limited to siRNAs.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides an illustration of a modified double stranded siRNA conjugated to a three saccharide ligand moiety modified with thiophosphate groups.

FIG. 2 is a bar chart illustrating the in vitro determination of TTR knockdown of TTR siRNA GalNAc conjugates STS016 L14 and L15. GN_Luc represents the negative control. mRNA level were normalised against PTEN.

FIG. 3 is a bar chart illustrating in vivo efficacy in mice with in vivo efficacy of TTR knockdown in mice. Mice (4 animals per group) were treated with a single subcutaneous dose of 1 mg/kg. Blood was taken after each timepoint (day 8, 15, 22 post injection) and analysed for TTR level using commercially available murineTTR specific Elisa Kit.

The present application is exemplified by the following examples:

EXAMPLES

General Information

All reactions were carried out under a nitrogen atmosphere, unless stated otherwise. NMR spectra were recorded on a Bruker 400 MHz Ultrashield™ and all chemical shifts (δ) were determined relative to TMS.

Example 1—Synthesis of GalNAc Phosphoramidites

(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyldiacetate (19)

To a solution of 15 (64.2 g, 166 mmol) in 1,2-dichloroethane (700 mL) was added trimethylsilyl trifluoromethanesulfonate (22.10 g, 99 mmol, 18.04 mL, 0.6 equiv) and the brown suspension was stirred for 15 minutes. Grinded 4A molecular sieves (85 g) were added and stirring was continued for 15 minutes. Triethyleneglycolmonobenzyl ether (51.8 g, 215 mmol, 47.5 mL, 1.3 equiv) was added, via drop wise addition, over a period of 15 minutes and stirring was continued at room temperature. The reaction mixture was filtered over a plug of kieselguhr followed by rinsing with warm dichloromethane. The filtrate was quenched by pouring in ice-cold aqueous saturated NaHCO3 solution (800 mL) and stirred vigerously. The layers were separated and the aqueous layer was extracted twice more with dichloromethane (2×300 mL). The combined organic layers were washed with water (600 mL) and brine (600 mL), dried over Na2SO4, filtered and concentrated in vacuo to obtain a yellow oil. Purification was performed by flash column chromatography (5-100% EtOAc in heptane) to obtain a mixture of 19 and triethyleneglycolmonobenzyl ether (64 g). This material was dissolved in dichloromethane (430 mL) followed by the addition of triethylamine (38.4 g, 380 mmol, 52.8 ml, 4 equiv) and DMAP (2.321 g, 19.00 mmol, 0.2 equiv). Then, via batch wise addition, was added TBDMSCl (21.47 g, 142 mmol, 1.5 equiv) and stirring was continued at room temperature for 2 hours. The reaction mixture was filtered and followed by pouring in an ice cold saturated solution of NaHCO3 (1 L). The layers were separated and the aqueous layer was extracted twice more with dichloromethane (2×300 mL). The combined organic layers were washed once with brine (1 L) and dried over Na2SO4. After concentrating in vacuo, followed by flash column chromatography (70-100% EtOAc in heptane), 19 was obtained as a colourless oil (36 g, yield 30%) 1H NMR (400 MHz, Chloroform-d) δ 7.38-7.28 (m, 5H), 6.58 (d, J=9.5 Hz, 1H), 5.26 (d, J=3.3 Hz, 1H), 4.96 (dd, J=11.2, 3.4 Hz, 1H), 4.79 (d, J=8.6 Hz, 1H), 4.53 (d, J=1.3 Hz, 2H), 4.28 (dt, J=11.2, 9.0 Hz, 1H), 4.16-4.06 (m, 2H), 3.88 (dd, J=6.0, 2.7 Hz, 2H), 3.75 (td, J=5.7, 2.7 Hz, 4H), 3.71-3.58 (m, 7H), 2.15 (s, 3H), 2.04 (s, 3H), 1.97 (s, 3H), 1.95 (s, 3H).

(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (ST20)

To a solution of 19 (47.68 g, 84 mmol) in tetrahydrofuran (330 ml) and 2-propanol (330 ml) was added 10% palladium on activated carbon (12.92 g, 12.14 mmol, 1.45 equiv). The reaction mixture was charged with hydrogen (balloon) and stirring was continued at room temperature overnight. The reaction mixture was filtered over kieselguhr and rinsed with warm dichloromethane. After concentrating in vacuo, ST20 was obtained (37 g, yield 94%) 1H NMR (400 MHz, DMSO-d6) δ 7.81 (d, J=9.2 Hz, 1H), 5.22 (d, J=3.3 Hz, 1H), 4.97 (dd, J=11.2, 3.4 Hz, 1H), 4.61 (t, J=5.4 Hz, 1H), 4.56 (d, J=8.4 Hz, 1H), 4.03 (s, 3H), 3.88 (dt, J=11.1, 8.9 Hz, 1H), 3.82-3.73 (m, 1H), 3.63-3.45 (m, 9H), 3.41 (t, J=5.1 Hz, 2H), 2.11 (s, 3H), 2.00 (s, 3H), 1.89 (s, 3H), 1.78 (s, 3H).

(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-(((2-cyanoethoxy)(diisopropylamino)phosphino)oxy)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (ST21)

To a solution of 4,5-dicyanoimidazole (961 mg, 8.13 mmol, 0.65 equiv) in anhydrous acetonitrile (8 mL) and dry dichloromethane (40 ml) were added grinded 4A molecular sieves (4.4 g). Then, 2-cyanoethyl tetraisopropylphosphoro-diamidite (4903 mg, 16.27 mmol, 5.16 ml, 1.3 equiv) was added via a syringe and stirred at room temperature for 10 minutes. Then, a solution of ST20 (6000 mg, 12.51 mmol) in dry dichloromethane (20 ml) was added to the reaction mixture over a period of 10 minutes. The reaction mixture was filtered over a cotton plug followed by concentrating in vacuo. Purification by flash column chromatography was performed twice (10-100% EtOAc in heptane) to obtain ST21 as a pale yellow oil (6.9 g, yield 74%). 1H NMR (400 MHz, DMSO-d6) δ 7.81 (d, J=9.3 Hz, 1H), 5.21 (d, J=3.4 Hz, 1H), 4.97 (dd, J=11.2, 3.4 Hz, 1H), 4.55 (d, J=8.5 Hz, 1H), 4.03 (d, J=3.0 Hz, 3H), 3.88 (dt, J=11.2, 8.9 Hz, 1H), 3.82-3.45 (m, 16H), 2.77 (t, J=6.1 Hz, 2H), 2.11 (s, 3H), 2.00 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H), 1.13 (dd, J=6.8, 3.1 Hz, 12H).

(3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazole-6,7-diyl diacetate (15)

To a suspension of Galactosamine pentaacetate (125 g, 321 mmol) in dichloromethane (870 mL) at room temperature was added, via drop wise addition, trimethylsilyltrifluoromethanesulfonate (107 g, 482 mmol, 87 mL, 1.5 equiv) over a period of 30 minutes. The reaction mixture was heated to 40° C. for a period of 2 hours, after which it was cooled back to room temperature and quenched by pouring in an ice-cold aqueous saturated NaHCO3 solution (1000 mL). The layers were separated and the aqueous layer was extracted twice more with dichloromethane (2×300 mL). The combined organic layers were washed with water (500 mL) and brine (800 mL), followed by drying over Na2SO4. After concentrating in vacuo 15 was obtained as a pale yellow oil (109 g, crude yield 103%). 1H NMR (400 MHz, Chloroform-d) δ 6.00 (d, J=6.8 Hz, 1H), 5.47 (t, J=3.0 Hz, 1H), 4.91 (dd, J=7.4, 3.3 Hz, 1H), 4.29-4.06 (m, 3H), 4.03-3.97 (m, 1H), 2.13 (s, 3H), 2.07 (d, J=1.0 Hz, 6H), 2.06 (d, J=1.3 Hz, 3H).

(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(4-(benzyloxy)butoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (16)

To a solution of 15 (109 g, 331 mmol) in dichloromethane (1200 mL) were added powdered molsieves 4A (75 g) followed by stirring for 15 minutes at room temperature. To the mixture was added 4-benzyloxy-1-butanol (89 g, 497 mmol, 87 mL, 1.5 equiv) and stirring was continued for another 15 minutes. Then, via dropwise addition, was added trimethylsilyltrifluoromethanesulfonate (44.1 g, 199 mmol, 36.0 mL, 0.6 equiv) over a period of 15 minutes. Stirring of the reaction mixture was continued for 2 hours. Filtration of the mixture was performed over a plug of kieselguhr followed by rinsing once with dichloromethane (200 mL). The filtrate was then quenched by pouring in an ice-cold saturated aqueous NaHCO3 solution (1000 mL). The layers were separated followed by extracting the aqueous layer twice more with dichloromethane (2×500 mL). The combined organic layers were washed with water (600 mL) and brine (600 mL) followed by drying over Na2SO4. After concentrating in vacuo, purification was performed by flash column chromatography on silica neutralized with 1% Et3N (20-80% EtOAc in heptane) to obtain 16 as a colourless oil which slowly crystalized (109 g, yield 65%). 1H NMR (400 MHz, DMSO-d6) δ 7.83 (d, J=9.3 Hz, 1H), 7.39-7.23 (m, 5H), 5.21 (d, J=3.5 Hz, 1H), 4.96 (dd, J=11.2, 3.5 Hz, 1H), 4.48 (d, J=8.5 Hz, 1H), 4.44 (s, 2H), 4.07-3.97 (m, 3H), 3.87 (dt, J=11.2, 8.8 Hz, 1H), 3.72 (p, J=5.3 Hz, 1H), 3.49-3.37 (m, 3H), 2.10 (s, 3H), 1.99 (s, 3H), 1.89 (s, 3H), 1.76 (s, 3H), 1.54 (qd, J=8.0, 5.2, 4.6 Hz, 4H).

(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(4-hydroxybutoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (17)

To a solution of 16 (109.6 g, 215 mmol) in tetrahydrofuran (1000 mL) and 2-propanol (1000 mL) was added 10% palladium on carbon (17.17 g, 16.13 mmol, 10%, 0.075 equiv) and the flask was charged with hydrogen (atmospheric pressure). Stirring of the reaction mixture was continued overnight at room temperature. The mixture was filtered over a plug of kieselguhr and concentrated in vacuo. After stripping the material twice with toluene (2×300 mL) and dichloromethane (2×300 mL), 17 was obtained as a white sticky solid (87 g, yield 97%). 1H NMR (400 MHz, Methanol-d4) δ 5.33 (dd, J=3.5, 1.0 Hz, 1H), 5.05 (dd, J=11.3, 3.3 Hz, 1H), 4.55 (d, J=8.5 Hz, 1H), 4.20-3.97 (m, 4H), 3.87 (dt, J=10.1, 5.8 Hz, 1H), 3.60-3.48 (m, 3H), 3.30 (p, J=1.8 Hz, 1H), 2.14 (s, 3H), 2.02 (s, 3H), 1.94 (s, 3H), 1.92 (s, 3H), 1.61 (dtd, J=16.8, 11.0, 10.1, 3.6 Hz, 4H).

(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(4-(((2-cyanoethoxy)(diisopropylamino)phosphino)oxy)butoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (ST23)

To a solution of 4,5-dicyanoimidazole (1.940 g, 16.43 mmol, 0.65 equiv) in dry acetonitrile (20 mL) and dry dichloromethane (20 mL), under an argon atmosphere, were added grinded Molsieves 4A (9 g). Then, 2-cyanoethyl tetraisopropylphosphoro-diamidite (10.00 g, 33.2 mmol, 10.53 mL, 1.31 equiv) was added via a syringe and stirred at room temperature for 10 minutes. Via drop wise addition was then added a solution of 17 (10.6 g, 25.3 mmol) in dry dichloromethane (50 mL) over a period of 10 minutes. After stirring for an additional 30 minutes, the reaction mixture was filtered over a cotton plug and concentrated in vacuo. Purification of the material was performed by multiple flash column chromatograph steps (0-100% EtOAc in heptane with 5% Et3N) to obtain ST23 as a pale yellow oil (11.75 g, yield 72%). 1H NMR (400 MHz, DMSO-d6) δ 7.82 (d, J=9.2 Hz, 1H), 5.21 (d, J=3.4 Hz, 1H), 4.96 (dd, J=11.2, 3.5 Hz, 1H), 4.48 (d, J=8.5 Hz, 1H), 4.02 (s, 3H), 3.93-3.82 (m, 1H), 3.78-3.65 (m, 3H), 3.64-3.49 (m, 4H), 3.48-3.40 (m, 1H), 2.76 (t, J=5.9 Hz, 2H), 2.11 (s, 3H), 2.00 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H), 1.62-1.46 (m, 4H), 1.13 (dd, J=6.8, 3.6 Hz, 12H). 31P NMR (162 MHz, Chloroform-d) 147 (d, J=8.6 Hz)

6-(benzyloxy)hexan-1-ol (18)

To a cooled and vigerously stirred suspension of sodium hydride (90 g, 2242 mmol, 3.5 equiv) in tetrahydrofuran (500 mL) was added, via dropwise addition, a solution of 1,6-hexanediol (265 g, 2242 mmol, 3.5 equiv) in tetrahydrofuran (1000 mL) over a period of one hour. After stirring for an additional 30 minutes, a solution of benzyl bromide (76 mL, 641 mmol, 1 equiv) in tetrahydrofuran (500 mL) was added over a period of 30 minutes. Upon complete addition, the reaction mixture was allowed to reach room temperature and stirring was continued overnight. The reaction mixture was cooled to a temperature of 5° C. followed by the slow addition of water (200 mL). The mixture was then concentrated in vacuo, redissolved in dichloromethane (600 mL) and washed with water (3000 mL). The aqueous layer was extracted three more times with dichloromethane (3×500 mL). The combined organic layers were washed with water (3×400 mL) and brine (1×500 mL) followed by drying over Na2SO4 and concentrating in vacuo. Purification was performed by gravity column chromatography (0-50% EtOAc in heptane) to obtain 18 (25 g, yield 20%). 1H NMR (400 MHz, Chloroform-d) δ 7.40-7.27 (m, 5H), 4.50 (s, 2H), 3.64 (t, J=6.7 Hz, 2H), 3.47 (t, J=6.6 Hz, 2H), 1.68-1.51 (m, 4H), 1.47-1.31 (m, 4H), 1.27 (s, 1H).

(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((6-(benzyloxy)hexyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (ST30Bn)

To a solution of 15 (28 g, 85 mmol) in dichloromethane (320 mL) were added powdered molsieves 4A (10 g) followed by stirring for 5 minutes. Then, 18 (26.6 g, 128 mmol, 1.5 equiv) was added and stirring was continued for another 15 minutes. Trimethylsilyl trifluoromethanesulfonate (9.26 mL, 51.0 mmol, 0.6 equiv) was added, via drop wise addition, over a period of 15 minutes. Stirring was continued at room temperature for 2 hours. The reaction mixture was filtered over a cotton plug followed by quenching with an ice-cold saturated aqueous NaHCO3 solution (300 mL). The layers were separated and extraction was performed twice more with dichloromethane (2×150 mL). The combined organic layers were washed with water (150 mL) and brine (150 mL) followed by drying over Na2SO4 and concentrating in vacuo. Purification was performed by flash column chromatography (20-100% EtOAc in heptane) to obtain ST30Bn as a colourless oil (25 g, yield 55%). 1H NMR (400 MHz, Chloroform-d) δ 7.38-7.27 (m, 5H), 5.43 (t, J=6.9 Hz, 1H), 5.38-5.27 (m, 2H), 4.71 (d, J=8.3 Hz, 1H), 4.50 (s, 2H), 4.21-4.07 (m, 2H), 3.96-3.81 (m, 3H), 3.47 (t, J=6.3 Hz, 3H), 2.14 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H), 1.94 (s, 3H), 1.68-1.51 (m, 4H), 1.44-1.30 (m, 4H).

(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((6-hydroxyhexyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (ST30)

To a solution of ST30Bn (29 g, 54 mmol) in tetrahydrofuran (250 mL) and 2-Propanol (250 mL) was added 10% palladium on carbon (0.582 g, 0.547 mmol, 0.075 equiv). The flask was charged with hydrogen (atmospheric pressure) and stirring of the mixture was continued overnight at room temperature. The reaction mixture was filtered over a plug of kieselguhr and the filtrate was concentrated in vacuo. After stripping twice with toluene (2×200 mL) and dichloromethane (2×200 mL) ST30 was obtained as a colourless oil (24 g, yield 99%). 1H NMR (400 MHz, Methanol-d4) δ 5.37-5.26 (m, 1H), 5.10-4.97 (m, 1H), 4.67-4.48 (m, 2H), 4.20-3.93 (m, 4H), 3.92-3.77 (m, 1H), 3.52 (hept, J=9.4, 8.1 Hz, 3H), 3.36-3.23 (m, 1H), 2.17-2.09 (m, 3H), 2.05-1.98 (m, 3H), 1.97-1.87 (m, 6H), 1.63-1.45 (m, 4H), 1.43-1.29 (m, 4H).

(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((6-(((2-cyanoethoxy)(diisopropylamino)phosphino)oxy)hexyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (ST31)

To a solution of ST30 (21.2 g, 47.4 mmol) in dry dichloromethane (550 mL), under an argon atmosphere, was added DIPEA (83 mL, 474 mmol, 10 equiv) and Molsieves 4A (30 g). The reaction mixture was cooled to a temperature of 0° C. followed by the drop wise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (13.46 g, 56.9 mmol, 1.2 equiv) over a period of 10 minutes. Stirring of the mixture was continued while allowing it to warm up over a period of 30 minutes. The reaction mixture was filtered over a cotton plug and directly coated on, with Et3N treated, silica (60 g). Purification was performed by flash column chromatography (10-60% EtOAc in heptane, 5% Et3N) to obtain ST31 as a yellow tar (24.8 g, yield 78%). 1H NMR (400 MHz, DMSO-d6) δ 7.82 (d, J=9.2 Hz, 1H), 5.21 (d, J=3.4 Hz, 1H), 4.96 (dd, J=11.3, 3.4 Hz, 1H), 4.48 (d, J=8.5 Hz, 1H), 4.02 (s, 3H), 3.91-3.81 (m, 1H), 3.79-3.63 (m, 3H), 3.63-3.49 (m, 4H), 3.45-3.37 (m, 1H), 2.76 (t, J=5.8 Hz, 2H), 2.10 (s, 3H), 2.00 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H), 1.58-1.40 (m, 4H), 1.37-1.22 (m, 4H), 1.13 (dd, J=6.8, 3.9 Hz, 12H).

Example 2—Synthesis of the Trebler Synthons

3,3′-((2-amino-2-((2-cyanoethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanenitrile (11)

To a vigerously stirred mixture of tris (50 g, 413 mmol) and 40% aqueous KOH (5.96 g, 42.5 mmol, 5.68 mL, 0.1 equiv) in 1,4-dioxane (100 mL) at 0° C. was added, via drop wise addition, acrylonitril (75 g, 1420 mmol, 94 mL, 3.44 equiv) over a period of 60 minutes. Upon complete addition the ice bath was removed and stirring of the mixture was continued overnight. To the mixture was added 1M aqueous HCl until pH=7. Additional water (100 mL) was added and the product was extracted with dichloromethane (2×200 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification was performed by gravity column chromatography (0-4% MeOH in dichloromethane) to obtain 11 as a yellow oil (78 g, yield 67%). 1H NMR (400 MHz, Chloroform-d) δ 3.70 (td, J=6.0, 2.5 Hz, 6H), 3.45 (d, J=2.8 Hz, 6H), 2.63 (qd, J=5.7, 1.8 Hz, 6H), 1.61 (s, 2H).

diethyl 3,3′-((2-amino-2-((3-ethoxy-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoate (12)

A solution of 11 (78 g, 278 mmol) in ethanol (400 mL) was bubbled through with hydrochloric acid gas for 15 minutes to obtain an opaque reaction mixture. this mixture was then refluxed overnight to observe the formation of ammonium chloride. The reaction mixture was cooled back to room temperature and bubbled through with nitrogen. Filtration was performed over a glass filter to remove the solids, followed by concentrating the filtrate in vacuo. The obtained brown oil was dissolved in dichloromethane (500 mL) and washed twice with aqueous saturated NaHCO3 solution (2×500 mL) and twice with water (2×500 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The obtained brown oil was redissolved in ethanol (400 mL), bubbled through with hydrochloric acid gas and refluxed overnight. The next day, the newly formed ammonium chloride was filtered off and the filtrate was concentrated in vacuo. The obtained yellow oil was dissolved in dichloromethane (500 mL) and washed twice with aqueous saturated NaHCO3 solution (2×500 mL) and twice with water (2×500 mL). to obtain 12 as a yellow oil (63 g, yield 54%). 1H NMR (400 MHz, Chloroform-d) δ 4.15 (q, J=7.1 Hz, 6H), 3.69 (t, J=6.4 Hz, 6H), 3.31 (s, 6H), 2.55 (t, J=6.4 Hz, 6H), 1.57 (s, 2H), 1.27 (t, J=7.2 Hz, 9H).

diethyl 3,3′-((2-(((benzyloxy)carbonyl)amino)-2-((3-ethoxy-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoate (13)

To a solution of 12 (63 g, 149 mmol) in 1,4-dioxane (630 mL) was added a solution of potassium carbonate (22.72 g, 164 mmol, 1.1 equiv) in water (63 mL). Then, the mixture was cooled to a temperature of 10° C. and via drop wise addition, over a period of 30 minutes, was added benzyl chloroformate (33.1 g, 194 mmol, 27.7 mL, 1.3 equiv). Stirring of the yellow suspension was continued overnight at room temperature. The reaction mixture was acidified to pH2 by the addition of 6M aqueous HCl. The white solids were removed by filtration over a glass filter and the filtrate was concentrated in vacuo. Purification was performed by gravity column chromatography (15-40% EtOAc in heptane) to obtain 13 as a pale yellow oil (68 g, yield 82%). 1H NMR (400 MHz, Chloroform-d) δ 7.38-7.28 (m, 5H), 5.26 (s, 1H), 5.04 (s, 2H), 4.13 (q, J=7.1 Hz, 6H), 3.75-3.60 (m 12H), 2.52 (t, J=6.3 Hz, 6H), 1.24 (t, J=7.1 Hz, 9H).

3,3′-((2-((3-hydroxypropoxy)methyl)-2-(methylamino)propane-1,3-diyl)bis(oxy))bis(propan-1-ol) (14)

A solution of 2.4 M lithium aluminium hydride in tetrahydrofuran (37.5 mL, 90 mmol, 10 equiv) in dry tetrahydrofuran (100 mL) was cooled to a temperature of −13° C. Then, to the reaction mixture was added a solution of 13 (5 g, 9.00 mmol, 1 equiv) in dry tetrahydrofuran (50 mL), via drop wise addition, over a period of one hour. Stirring of the reaction mixture was continued overnight while allowing it to slowly reach room temperature. Upon cooling, the reaction was quenched by the slow addition of water (3.4 mL), 4M aqueous NaOH (3.4 mL) and water (10 mL). The white precipitate was removed by filtration over a dry Na2SO4 plug followed by rinsing twice with tetrahydrofuran. The filtrate was concentrated in vacuo to obtain crude 14 as a pale yellow oil (2.92 g, yield corrected for purity 810%). 1H NMR (400 MHz, Chloroform-d) δ 3.78-3.70 (m, 6H), 3.63 (q, J=5.8 Hz, 6H), 3.42 (s, 6H), 2.91 (br, 3H), 2.35 (s, 3H), 1.80 (p, J=5.4 Hz, 6H), 1.72-1.66 (m, 1H).

9,9-bis((3-hydroxypropoxy)methyl)-2,2,3,3,8-pentamethyl-4,11-dioxa-8-aza-3-silatetradecan-14-ol (ST47)

To a solution of 14 (2.9 g, 7.22 mmol) and 3-(tert-Butyldimethylsiloxy)propionaldehyde (1.863 mL, 7.94 mmol, 1.1 equiv) in dichloromethane (58 mL) was added sodium triacetoxyborohydride (2.294 g, 10.83 mmol, 1.5 equiv). Stirring was continued for 1.5 hours at room temperature, followed by pouring the reaction mixture in a saturated aqueous solution of NaHCO3 (25 mL). The layers were separated and the aqueous layer was extracted once more with dichloromethane (25 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification of the crude material was performed by flash column chromatography (0-10% MeOH in dichloromethane) to obtain ST47 as a pale yellow oil (1.27 g, yield 33%). 1H NMR (400 MHz, Chloroform-d) δ 3.75 (t, J=5.4 Hz, 6H), 3.66-3.56 (m, 8H), 3.50 (s, 6H), 3.11 (br s, 3H), 2.64 (t, J=7.3 Hz, 2H), 2.33 (s, 3H), 1.80 (p, J=5.4 Hz, 6H), 1.64 (p, J=6.3 Hz, 2H), 0.90 (s, 9H), 0.05 (s, 6H).

8-((3-(bis(4-methoxyphenyl)(phenyl)methoxy)propoxy)methyl)-N-(3-((tert-butyldimethylsilyl)oxy)propyl)-1,1,15,15-tetrakis(4-methoxyphenyl)-N-methyl-1,15-diphenyl-2,6,10,14-tetraoxapentadecan-8-amine (ST47DMTrTBDMS)

Residual water was removed from ST47 (1.27 g, 2.42 mmol) by stripping twice with pyridine, followed by redissolving in pyridine (60 mL) under an argon atmosphere. To the reaction mixture were added molecular sieves 4A (4 g) and stirring was continued for 15 minutes. Solid DMTrCl (4.16 g, 12.28 mmol, 5 equiv) was added in batches over a period of 15 minutes. Stirring of the now dark orange mixture was continued overnight at room temperature. The reaction was quenched by the addition of MeOH (4.4 mL, 109 mmol, 45 equiv) and it was stirred for 5 minutes. Then, the reaction mixture was filtered over a cotton plug and the filtrate was coated on, with Et3N neutralized, silica (10 g). Purification was performed by flash column chromatography (0-100% EtOAc in heptane, 5% Et3N) to obtain ST47DMTrTBDMS as a yellow foaming oil (2.37 g, yield 52%). 1H NMR (400 MHz, Chloroform-d) δ 7.43-7.37 (m, 6H), 7.32-7.21 (m, 18H), 7.19-7.13 (m, 3H), 6.81-6.76 (m, 12H), 3.75 (s, 18H), 3.54 (q, J=6.2, 5.4 Hz, 2H), 3.39 (t, J=6.5 Hz, 6H), 3.32 (s, 6H), 3.07 (t, J=6.5 Hz, 6H), 2.51 (t, J=7.4 Hz, 2H), 2.19 (s, 3H), 1.80 (p, J=6.4 Hz, 6H), 1.62-1.52 (m, 2H), 0.86 (s, 9H), 0.01 (s, 6H).

3-((8-((3-(bis(4-methoxyphenyl)(phenyl)methoxy)propoxy)methyl)-1,1,15,15-tetrakis(4-methoxyphenyl)-1,15-diphenyl-2,6,10,14-tetraoxapentadecan-8-yl)(methyl)amino)propan-1-ol (ST47DMTrOH)

To a solution of ST47DMTrTBDMS (2.37 g, 1.280 mmol) in dry tetrahydrofuran (20 mL) was added a 1.0M TBAF solution in THF (1.433 mL, 1.433 mmol, 1.17 equiv) and the solution was stirred overnight. The material was coated on, with Et3N treated, silica and purification was performed by flash column chromatography (0-100% EtOAc in heptane) to obtain ST47DMTrOH as a white solid (1.37 g, yield 84%). 1H NMR (400 MHz, Chloroform-d) δ 7.43-7.38 (m, 6H), 7.32-7.22 (m, 18H), 7.19-7.14 (m, 3H), 6.82-6.76 (m, 12H), 3.75 (s, 18H), 3.63 (t, J=4.9 Hz, 2H), 3.38 (t, J=6.4 Hz, 6H), 3.31 (s, 6H), 3.07 (t, J=6.4 Hz, 6H), 2.66 (t, 2H), 2.15 (s, 3H), 1.80 (p, J=6.3 Hz, 6H), 1.58-1.46 (m, 3H).

3-((8-((3-(bis(4-methoxyphenyl)(phenyl)methoxy)propoxy)methyl)-1,1,15,15-tetrakis(4-methoxyphenyl)-1,15-diphenyl-2,6,10,14-tetraoxapentadecan-8-yl)(methyl)amino)propyl (2-cyanoethyl) diisopropylphosphoramidite (ST48)

To a solution of ST47DMtrOH (1.37 g, 1.075 mmol) in dry dichloromethane (20 mL) was added DIPEA (1.87 mL, 10.75 mmol, 10 equiv) and molecular sieves 4A (4 g) followed by cooling to a temperature of 0° C. Then, to the reaction was added 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.305 g, 1.290 mmol, 1.2 equiv) via drop wise addition over a period of 5 minutes. Stirring of the reaction mixture was continued for another 15 minutes while allowing it to reach room temperature. The reaction mixture was filtered over a cotton plug and the filtrate was coated on, with Et3N treated, silica (6 g). Purification was performed by flash column chromatography (0-100% EtOAc in heptane, 5% Et3N) to obtain ST48 as a colourless tar (1.27 g, yield 80%). 1H NMR (400 MHz, Chloroform-d) δ 7.43-7.37 (m, 6H), 7.33-7.21 (m, 18H), 7.20-7.13 (m, 3H), 6.82-6.75 (m, 12H), 3.83-3.68 (m, 20H), 3.66-3.48 (m, 4H), 3.38 (t, J=6.5 Hz, 6H), 3.31 (s, 6H), 3.07 (t, J=6.4 Hz, 6H), 2.60-2.48 (m, 4H), 2.19 (s, 3H), 1.80 (p, J=6.5 Hz, 6H), 1.66 (p, J=6.9 Hz, 2H), 1.15 (t, J=7.1 Hz, 12H).

di-tert-butyl 4-(3-(tert-butoxy)-3-oxopropyl)-4-((3-((tert-butyldimethylsilyl) oxy)propyl)amino)heptanedioate (5)

To a solution of aminotriester (1 g, 2.406 mmol) in dichloromethane (10 ml) was added 3-(tert-Butyldimethylsiloxy)propionaldehyde (0.565 ml, 2.406 mmol) and stirring was continued for 45 minutes. Then, to the reaction mixture was added sodium triacetoxyborohydride (1.530 g, 7.22 mmol) and stirring of the reaction mixture was continued at room temperature for 2 hours. The reaction mixture was washed with an aqueous saturated NaHCO3 solution (10 mL). the organic layer was filtered over a phase separator and the filtrate was concentrated in vacuo. Purification was performed by flash column chromatography (0-20% EtOAc in heptane) to obtain 5 as a colourless oil (1.07 g, 76% yield). 1H NMR (400 MHz, Chloroform-d) δ 3.68 (t, J=6.1 Hz, 2H), 2.50 (t, J=6.6 Hz, 2H), 2.23-2.10 (m, 6H), 1.66-1.50 (m, 8H), 1.44 (s, 27H), 0.89 (s, 9H), 0.05 (s, 6H).

di-tert-butyl 4-(3-(tert-butoxy)-3-oxopropyl)-4-((3-((tert-butyldimethylsilyl)oxy)propyl)(methyl)amino)heptanedioate (6)

A solution of 5 (1 g, 1.701 mmol), acetic acid (0.097 ml, 1.701 mmol) and formaldehyde 37% in water (1.266 ml, 17.01 mmol) in methanol (10 ml) was stirred for 30 minutes at room temperature. Then, sodium cyanoborohydride (0.321 g, 5.10 mmol) was added and stirring was continued for 2 hours. To the reaction was added a saturated solution of Na2CO3 (10 mL) and this was stirred vigerously. Additional water was added (15 mL) and the aqueous layer was extracted twice with EtOAc (20 mL). The combined organic layers were washed with brine (20 ml) and dried over Na2SO4. After concentrating in vacuo 6 was obtained (1.00 g, 98% yield). 1H NMR (400 MHz, Chloroform-d) δ 3.62 (t, J=6.6 Hz, 2H), 2.54 (t, J=7.2 Hz, 2H), 2.28-2.15 (m, 9H), 1.78-1.55 (m, 8H), 1.44 (s, 28H), 0.89 (s, 9H), 0.05 (s, 6H).

4-((3-((tert-butyldimethylsilyl)oxy)propyl)(methyl)amino)-4-(3-hydroxypropyl)heptane-1,7-diol (7)

To a cooled solution of 2.4 M lithium aluminium hydride in THF (0.757 g, 19.94 mmol) in dry tetrahydrofuran (20 ml) was added a solution of 6 (1 g, 1.661 mmol) in dry tetrahydrofuran (10 ml) via drop wise addition. The reaction mixture was allowed to warm up to room temperature and stirring was continued overnight. The reaction mixture was cooled to a temperature of −11° C. followed by the drop wise addition of water (0.76 mL), 4M NaOH (0.76 mL) and additional water (2.3 mL). The obtained white suspension was filtered over a kieselguhr plug and concentrated in vacuo to obtain 7 as a pale yellow oil (677 mg, 85% purity, 88% yield). 1H NMR (400 MHz, Chloroform-d) δ 3.67-3.53 (m, 8H), 2.71 (t, J=7.9 Hz, 2H), 2.36 (s, 3H), 1.76-1.50 (m, 14H), 1.28 (s, 3H), 0.89 (s, 9H), 0.05 (s, 6H).

1,7-bis(bis(4-methoxyphenyl)(phenyl)methoxy)-4-(3-(bis(4-methoxyphenyl) (phenyl)methoxy)propyl)-N-(3-((tert-butyldimethylsilyl)oxy)propyl)-N-methylheptan-4-amine (ST49)

7 (0.677 g, 1.729 mmol) was stripped twice with pyridine, followed by redissolving in pyridine (7 ml) and placing under a nitrogen atmosphere. Then, to the reaction mixture was added DMTrCl (2.64 g, 7.78 mmol) via batch wise addition to obtain a dark red solution. Stirring was continued overnight at room temperature to obtain a brown suspension. The material was coated on, with Et3N neutralized, silica and purification was performed by flash column chromatography (0-40% EtOAc in heptane both treated with 5% Et3N) to obtain ST49 as a yellow foaming solid (1.4 g, 55% yield). 1H NMR (400 MHz, Chloroform-d) δ 7.47-7.40 (m, 6H), 7.37-7.21 (m, 18H), 7.20-7.13 (m, 3H), 6.82-6.74 (m, 12H), 3.75 (s, 18H), 3.61 (t, J=6.5 Hz, 2H), 3.00 (t, J=6.4 Hz, 6H), 2.54 (t, J=7.1 Hz, 2H), 2.23 (s, 3H), 1.67-1.52 (m, 8H), 1.46-1.33 (m, 6H), 0.84 (s, 9H), 0.01 (s, 6H).

3-((1,7-bis(bis(4-methoxyphenyl)(phenyl)methoxy)-4-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)heptan-4-yl)(methyl)amino)propan-1-ol (ST490H)

To a solution of ST49 (1.4 g, 0.949 mmol) in dry tetrahydrofuran (23 ml) was added 1M TBAF in THF (1.613 ml, 1.613 mmol) via a single stream and stirring of the orange solution was continued overnight. The reaction mixture was coated on, with Et3N neutralized, silica and purification was performed by flash column chromatography (0-70% EtOAc in heptane both treated with 5% Et3N) to obtain ST490H as a white foam (853 mg, 73% yield). 1H NMR (400 MHz, Chloroform-d) δ 7.46-7.38 (m, 6H), 7.36-7.20 (m, 18H), 7.20-7.13 (m, 3H), 6.82-6.74 (m, 12H), 3.82-3.70 (m, 20H), 3.03 (t, J=5.9 Hz, 6H), 2.80 (t, J=5.4 Hz, 2H), 2.28 (s, 3H), 1.73-1.45 (m, 15H).

3-((1,7-bis(bis(4-methoxyphenyl)(phenyl)methoxy)-4-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)heptan-4-yl)(methyl)amino)propyl (2-cyanoethyl) diisopropylphosphoramidite (ST50)

ST490H (852 mg, 0.719 mmol) was dissolved in dry dichloromethane (10 mL) followed by the addition of 4A molecular sieves (1.7 g) and DIPEA (1.256 ml, 7.19 mmol). The mixture was cooled to a temperature of −15° C. and to this was added 2-CyanoethylN,N-diisopropylchlorophosphoramidite (203 mg, 0.856 mmol) via drop wise addition over a period of 10 minutes. Stirring of the reaction mixture was continued while allowing it to reach room temperature. The reaction mixture was filtered over a cotton plug followed by coating on, with ET3N treated, silica. Purification was performed by flash column chromatography (0-40% EtOAc in heptane both treated with 5% Et3N) to obtain ST50 as a pale yellow tar (624 mg, 62% yield). 1H NMR (400 MHz, Chloroform-d) δ 7.47-7.40 (m, 6H), 7.35-7.21 (m, 18H), 7.21-7.13 (m, 3H), 6.83-6.74 (m, 12H), 3.74 (s, 21H), 3.62-3.49 (m, 3H), 3.00 (t, J=6.3 Hz, 6H), 2.65-2.43 (m, 4H), 2.23 (s, 3H), 1.77-1.67 (m, 2H), 1.63-1.50 (m, 6H), 1.49-1.33 (m, 6H), 1.13 (dd, J=18.3, 6.7 Hz, 12H).

Example 3—Synthesis of Nucleic Acid Conjugates

All Oligonucleotides were synthesized on an AKTA oligopilot synthesizer. Commercially available solid support and 2′O-Methyl RNA phosphoramidites, 2′Fluoro, 2′Deoxy RNA phosphoramidites and commercially available long trebler phosphoramidite (STKS) (Glen research) were used. Oligonucleotide synthesis, deprotection and purification followed standard procedures that are known in the art. Oligonucleotide and oligonucleotide conjugate synthesis was performed by a commercial oligonucleotide manufacturer (Biospring, Frankfurt, Germany).

Conjugation of the GalNAc synthons (ST21, ST23, ST31) or trebler synthons (STKS, ST48, ST50) was achieved by coupling of the respective phosphoramidite to the 5′ end of the oligochain under standard phosphoramidite coupling conditions. Phosphorothioates were introduced using standard commercially available thiolation reagents (EDITH, Link technologies).

The single strands were cleaved off the CPG by using aqueous Methylamine and the resulting crude oligonucleotide was purified by Ionexchange chromatography (Resource Q, 6 mL, GE Healthcare) on a AKTA Pure HPLC System using a Sodium chloride gradient. Product containing fractions were pooled, desalted on a size exclusion column (Zetadex, EMP Biotech) and lyophilised.

For Duplexation, equimolar amounts of the respective single strands were dissolved in water and heated to 80° C. for 5 min. After cooling the resulting Duplex was lyophilised.

Sequences

Modifications key for the following sequences:

f denotes 2′Fluoro 2′deoxyribonucleotide

m denotes 2′O Methyl ribonucleotide

(ps) denotes phosphorothioate linkage

STS016 L14 Antisense strand: 5′ mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG (ps) fU (ps) mU 3′ Sense strand: (ST23 (ps))3 ST48 (ps) fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU (ps) mA (ps) fA STS016 L15 Antisense strand: 5′ mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG (ps) fU (ps) mU 3′ Sense strand: (ST31 (ps))3 ST48 (ps) fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU (ps) mA (ps) fA GN_Luc (non targeting control) Antisense strand: 5′mU (ps)fC(ps)mGfAmAfGmUfAmUfUmCfCm GfCmGfUmA (ps) fC(ps)mG 3′ Sense strand: 5′(ST23(ps))3 STKS(ps)fCmGfUmAfCmGfCmG fGmAfAmUfAmCfUmUfC (ps)mG(ps)fA 3′

TABLE 2 Mass spectrometry data for oligonucleotides Oligonucleotide MS found MS calculated antisense strand (STS016 L14 6943 Da 6943.33 Da and L15) STS016 L14 sense strand 8372 Da 8375.52 Da STS016 L15 sense strand 8456 Da 8459.52 Da GN_Luc antisense 6260 Da 6259.93 Da GN_Luc sense 7799 Da 7800.21 Da

Example 4—In Vitro Determination of TTR Knockdown of Various TTR siRNA GalNAc Conjugates

In vitro determination of TTR knockdown of various TTR siRNA GalNAc conjugates STS016 L14 and L15 was determined in a hepatocyte assay.

Primary Hepatocytes (Life technology) were seeded into 6 well plates (600,000 cells per well) according to manufacturer's protocol and incubated with the respective concentration of the GalNAc conjugate. Cells were harvested 24 h post incubation and RNA was isolated and analysed using Taqman analysis as described below:

Target Gene Expression In Vitro:

25-100 ng total RNA was used for quantitative TaqMan RT-PCR with the amplicon sets obtained from BioTez GmBH, Berlin, Germany: The TaqMan RT-PCR reactions were carried out with an ABI PRISM 7700 Sequence Detector (Software: Sequence Detection System v1.6.3 (ABI Life Technologies)) or StepOnePlus Real Time PCR System (ABI) using a standard protocol for RT-PCR as described previously (Fehring et al.) 5 with primers and probes at a concentration of 300 and 100 nmol/l respectively. TaqMan data were calculated by using the comparative Ct method. mRNA level were normalised against PTEN.

Amplicon sets for detection of TTR mRNA mmTTR:467U22 TGGACACCAAATCGTACTGGAA mmTTR:550L22 CAGAGTCGTTGGCTGTGAAAAC mmTTR:492U27FL ACTTGGCATTTCCCCGTTCCATGAATT Amplicon sets for detection of PTEN mRNA PTEN CACCGCCAAATTTAACTGCAGA PTEN AAGGGTTTGATAAGTTCTAGCTGT PTEN TGCACAGTATCCTTTTGAAGACCATAACCCA

Both siRNA GalNAc conjugates L14 and L15 were very effective in reducing TTR levels. Results are shown in FIG. 2.

Example 5—In Vivo Assay and Duration of TTR Knockdown in Mice

8 weeks old male C57BL/6JOlaHsd mice were injected with the dose of 1 mg/kg with a single subcutaneous Injection of 300 uL/kg (4 animals per group). PBS was used as control.

Blood was taken after each timepoint (day 8, 15, 22 post injection) and analysed for TTR level using commercially available murineTTR specific Elisa Kit.

All different siRNA GalNAc conjugates STS016 L14 and STS016 L15 were very effective in reducing TTR levels. Results are shown in FIG. 3.

REFERENCES

  • 1. Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998, 391 (6669), 806-11.
  • 2. Elbashir, S. M.; Lendeckel, W.; Tuschl, T., RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes &development 2001, 15 (2), 188-200.
  • 3. Dubber, M.; Frechet, J. M., Solid-phase synthesis of multivalent glycoconjugates on a DNA synthesizer. Bioconjugate chemistry 2003, 14 (1), 239-46.
  • 4. Weigel, P. H.; Yik, J. H., Glycans as endocytosis signals: the cases of the asialoglycoprotein and hyaluronan/chondroitin sulfate receptors. Biochim Biophys Acta 2002, 1572 (2-3), 341-63.
  • 5. Ishibashi, S.; Hammer, R. E.; Herz, J., Asialoglycoprotein receptor deficiency in mice lacking the minor receptor subunit. J Biol Chem 1994, 269 (45), 27803-6.
  • 6. Biessen, E. A.; Broxterman, H.; van Boom, J. H.; van Berkel, T. J., The cholesterol derivative of a triantennary galactoside with high affinity for hepatic asialoglycoprotein receptor: a potent cholesterol lowering agent. J Med Chem 1995, 38 (11), 1846-52.
  • 7. Akinc, A.; Querbes, W.; De, S.; Qin, J.; Frank-Kamenetsky, M.; Jayaprakash, K. N.; Jayaraman, M.; Rajeev, K. G.; Cantley, W. L.; Dorkin, J. R.; Butler, J. S.; Qin, L.; Racie, T.; Sprague, A.; Fava, E.; Zeigerer, A.; Hope, M. J.; Zerial, M.; Sah, D. W.; Fitzgerald, K.; Tracy, M. A.; Manoharan, M.; Koteliansky, V.; Fougerolles, A. d.; Maier, M. A., Targeted Delivery of RNAi Therapeutics With Endogenous and Exogenous Ligand-Based Mechanisms. Molecular therapy: the journal of the American Society of Gene Therapy 2010, 18 (7), 1357-1364.
  • 8. Fehring, V.; Schaeper, U.; Ahrens, K.; Santel, A.; Keil, O.; Eisermann, M.; Giese, K.; Kaufnann, J., Delivery of therapeutic siRNA to the lung endothelium via novel Lipoplex formulation DACC. Mol Ther 2014, 22 (4), 811-20.
  • 9. Prakash, T. P.; Brad Wan, W.; Low, A.; Yu, J.; Chappell, A. E.; Gaus, H.; Kinberger, G. A.; Østergaard, M. E.; Migawa, M. T.; Swayze, E. E.; Seth, P. P., Solid-phase synthesis of 5′-triantennary N-acetylgalactosamine conjugated antisense oligonucleotides using phosphoramidite chemistry. Bioorganic & medicinal chemistry letters 2015, 25 (19), 4127-4130.
  • 10. Li, L.-C.; Okino, S. T.; Zhao, H.; Pookot, D.; Place, R. F.; Urakami, S.; Enokida, H.; Dahiya, R., Small dsRNAs induce transcriptional activation in human cells. Proceedings of the National Academy of Sciences 2006, 103 (46), 17337-17342.

Claims

1. A compound having the formula I:

[S—X1—P—X2]3-A-X3—Z  (I)
wherein:
S represents a saccharide;
X1 represents C3-C6 alkylene or (—CH2—CH2—O)m(—CH2)2— wherein m is 1, 2, or 3;
P is a modified phosphate;
X2 is C1-C8 alkylene;
X3 represents a bridging unit;
Z is a nucleic acid;
the linkage between X3 and Z is a phosphate or thiophosphate;
and A is a branching unit selected from:
wherein:
R1 is hydrogen or C1-C10 alkylene;
and R2 is C1-C10 alkylene.

2. The compound of claim 1, wherein A has the structure:

wherein:
R1 is hydrogen or C1-C10 alkylene; and
R2 is C1-C10 alkylene.

3. The compound of claim 2, wherein A has a structure selected from:

wherein in each case
X3 is attached to the nitrogen atom.

4. The compound of claim 1, wherein X3 may be selected from —C1-C20 alkylene-, —C2-C20 alkenylene-, an alkylene ether of formula —(C1-C20 alkylene)-O—(C1-C20 alkylene)-, —C0-C4 alkylene(Cy)C0-C4 alkylene- wherein Cy represents a substituted or unsubstituted 5 or 6 membered cycloalkylene, arylene, heterocyclylene or heteroarylene ring, —C1-C4 alkylene-NHC(O)—C1-C4 alkylene-, —C1-C4 alkylene-C(O)NH—C1-C4 alkylene-, —C1-C4 alkylene-SC(O)—C1-C4 alkylene-, —C1-C4 alkylene-C(O)S—C1-C4 alkylene-, —C1-C4 alkylene-OC(O)—C1-C4 alkylene-, —C1-C4 alkylene-C(O)O—C1-C4 alkylene-, and —C1-C6 alkylene-S—S—C1-C6 alkylene-.

5. The compound of claim 4, wherein X3 is:

a. —C1-C20 alkylene-;
b. selected from the group consisting of —C3H6—, —C6H12— and —C8H16—; or,
c. —C3H6—.

6. The compound of claim 1, wherein the modified phosphate is a thiophosphate.

7. The compound of claim 1, wherein the saccharide is:

a. selected from N-acetyl galactosamine, mannose, galactose, glucose, glucosamine and fructose; or
b. N-acetyl galactosamine.

8. The compound of claim 1, wherein X1 is:

a. (—CH2—CH2—O)m(—CH2)2— wherein m is 1, 2, or 3; or
b. C3-C6 alkylene.

9. The compound of claim 1, wherein the compound has one of the following formulas:

wherein Z is a nucleic acid.

10. The compound of claim 1, wherein the nucleic acid is selected from RNAi, siRNA, siNA, antisense nucleic acid, ribozymes, aptamers and spiegelmers.

11. The compound of claim 1, wherein the nucleic acid is modified.

12. The compound of claim 11 wherein the modification is selected from substitutions or insertions with analogues of nucleic acids or bases and chemical modification of the base, sugar or phosphate moieties.

13. A composition comprising a compound as defined in claim 1 and a pharmaceutically suitable carrier or excipient.

14. A method for treating a subject in need thereof, comprising a step of administering the compound of claim 1 to the subject.

15. The method of claim 14, wherein the subject is suffering from a liver disease, a genetic disease, hemophilia or bleeding disorder, liver fibrosis, non-alcoholic steotohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), viral hepatitis, a rare disease, a metabolic disease, a cardiovascular disease, obesity, thalassemia, liver injury, hemochromatosis, alcoholic liver disease, alcohol dependence, anemia, or anemia of chronic disease.

16. A method of delivering nucleic acids to a hepatocyte, the method comprising contacting the hepatocyte with a compound of claim 1.

17. The method of claim 15, wherein the rare disease is acromegaly; the metabolic disease is hypercholesterolemia, dyslipidemia, or hypertriglyceridemia; or the liver injury is drug induced liver injury.

18. A method of treating a subject in need thereof, the method comprising a step of administering a composition of claim 10 to the subject.

19. The method of claim 17, wherein the subject is suffering from a liver disease, a genetic disease, hemophilia or bleeding disorder, liver fibrosis, non-alcoholic steotohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), viral hepatitis, a rare diseases, a metabolic diseases, a cardiovascular disease, obesity, thalassemia, liver injury, hemochromatosis, alcoholic liver disease, alcohol dependence, anemia, or anemia of chronic diseases.

20. The method of claim 19, wherein the rare disease is acromegaly; the metabolic disease is hypercholesterolemia, dyslipidemia, or hypertriglyceridemia; or the liver injury is drug induced liver injury.

21. The compound of claim 10, wherein the nucleic acid is a siRNA and wherein the siRNA comprises:

a. 1-5 2′-O-CH3 modified nucleotides;
b. 5-10 2′-O-CH3 modified nucleotides;
c. 15-20 2′-O-CH3 modified nucleotides;
d. 20-25 2′-O-CH3 modified nucleotides; or
e. 25-30 2′-O-CH3 modified nucleotides.

22. The compound of claim 10, wherein the nucleic acid is a siRNA and wherein the siRNA comprises an antisense strand of 19 nucleotides in length and a sense strand 19 nucleotides in length, wherein said antisense strand comprises 2′-O-CH3 modifications at nucleotides 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, and wherein said sense strand comprises 2′-O-CH3 modifications at nucleotides 2, 4, 6, 8, 10, 12,14, 16 and 18, wherein said antisense strand is numbered from 5′-3′ and said sense strand is numbered from 3′-5′.

23. The compound of claim 10, wherein the nucleic acid is a siRNA, wherein the siRNA is blunt-ended on both ends and wherein the siRNA has a length of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 consecutive nucleotides.

Referenced Cited
U.S. Patent Documents
5885968 March 23, 1999 Biessen et al.
Foreign Patent Documents
0586520 March 1994 EP
0618925 October 1994 EP
1992/20823 November 1992 WO
1993/13121 July 1993 WO
1996/10390 April 1996 WO
1996/10391 April 1996 WO
1996/10392 April 1996 WO
1999/54459 October 1999 WO
2014/179620 November 2014 WO
2016/055601 April 2016 WO
2017/174657 October 2017 WO
Other references
  • Akinc et al., Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms, Molecular Therapy, 18(7):1357-1364 (2010).
  • Biessen et al., The cholesterol derivative of a triantennary galactoside with high affinity for hepatic asialoglycoprotein receptor: a potent cholesterol lowering agent, J. Med. Chem., 38(11):1846-52 (1995).
  • Dubber et al., Solid-phase synthesis of multivalent glycoconjugates on a DNA synthesizer, Bioconjugate Chemistry, 14(1):239-46 (2003).
  • Elbashir et al., RNA interference is mediated by 21-and 22-nucleotide RNAs, Genes & Development, 15(2):188-200 (2001).
  • Fehring et al., Delivery of therapeutic siRNA to the lung endothelium via novel Lipoplex formulation DACC, Mol. Ther., 22(4):811-20 (2014).
  • Fire et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature, 391 (6669):806-11 (1998).
  • International Application No. PCT/EP2018/058708, International Preliminary Report on Patentability, dated Oct. 17, 2019.
  • International Application No. PCT/EP2018/058708, International Search Report and Written Opinion, dated May 18, 2018.
  • Ishibashi et al., Asialoglycoprotein receptor deficiency in mice lacking the minor receptor subunit, J. Biol. Chem., 269(45):27803-6 (1994).
  • Li et al., Small dsRNAs induce transcriptional activation in human cells, Proceedings of the National Academy of Sciences, 103(46):17337-17342 (2006).
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Patent History
Patent number: 11299738
Type: Grant
Filed: Apr 5, 2018
Date of Patent: Apr 12, 2022
Patent Publication Number: 20210087570
Assignee: SILENCE THERAPEUTICS GMBH (Berlin)
Inventors: Dmitry Samarsky (Berlin), Christian Frauendorf (Berlin)
Primary Examiner: Sean McGarry
Application Number: 16/603,211
Classifications
Current U.S. Class: Non/e
International Classification: C12N 15/113 (20100101); C12Q 1/68 (20180101);